Genetics and Biochemistry of Secondary Metabolism

Genetics and Biochemistry of Secondary Metabolism VEDPAL SINGHMALIK] The Upjohn Company. Kalamazoo. Michigan

28 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 I1. Illegitimate Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . 32 111. Enzymes of Secondary Metabolism . . . . . . . . ........ 34 A . Inducible Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Compartmentalization . . . . . . . . . . . . . . . . . . . . . . . . . 35 36 C . Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 IV . Controlling Effect of the Environment . . . . . . . . . . . . . . . . . . . . 38 A . Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 B . Trace Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 C . Hydrogen Ion Concentration . . . . . . . . . . 39 D . Temperature and Aeration . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 V. Genetics of Secondary Metabolism. . . . . . . . . . . . . . . . . . . . . . . . 41 A . Chromosomal Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 B . Extrachromosomal Elements . . . . . . . . . . . . . . . . . . . . . . . . . 48 C . Genetic Engineering of Secondary Metabolites . . . . . . . . . 51 D . Reciprocal Genetics of Secondary Metabolism . . . . . . . . . . VI . Control of Secondary Metabolism . . ........ 53 55 A . Growth-Linked Suppression . . . . . . . . . . . . . . . . . . . . . . . . . 58 B. Multivalent Induction by Precursors . . . . . . . . . . . . . . . . . . 74 C . Feedback Inhibition and End Product Repression. . . . . . . 77 D . Catabolite Repression 81 E . Enzyme Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 F . Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 G . Glutamine Synthetase . . . . . . . . . . . . . . . . . . . . . . . . 85 H . Energy Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 VII . Regulation of Autotoxicity . . . . . . . . . . . 87 A . Regulation of Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 B . Modification of Target Site . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 C . Biotransformation and Regulation of Catabolism . . . . . . . . VIII . Secondary Metabolism, S 94 Exoenzyme Formation . . 97 IX . Role of Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 X . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

'Present address: Philip Morris Research Center. P.O. Box 26583. Richmond. Virginia 23261.

27 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 28 Copyright @ 1982 by Academic Press. Ioc . All rights of reproduction 111 any form reserved. ISBN 0-12-W2628-7

28

VEDPAL SINGH MALIK

I. Introduction One must know all the literature, but not trust any of it.

RICHARDWILLSTATTER

In his classic text on the chemical activities of fungi, the great American microbiologist J. W. Foster (1949) conveyed the essence of secondary metabolism as perceived at that time: It would appear that the enzyme mechanisms usually involved in complete oxidation of the substrate Iwcome saturated, and the snbstrate molecules then are excreted and accumulate as such, or thcy are shunted to secondary or subsidiary enzyme systems which are able to affect only relatively ininor changes in the substance, which then accumulates in its transformed state.

Studies of the biosynthetic pathways of secondary metabolites have since shown that chemical changes of the starter substrate are much more complex than suggested by Foster (Queener et aZ., 1979; Martin and Demain, 1978). As a matter of fact, the synthesis of secondary metabolites is carried out by specific pathways involving enzymes coded by genes that are not involved in growth. Plant physiologists were the first to recognize that certain compounds, such as alkaloids, terpenes, camphor, and tannins, were obtained only from particular plant species. The distribution of these compounds was not universal among the plant kingdom, and they could not be assigned a specific fimction. These metabolites were dubbed as secondary products of metabolism (Ruhland, 1958). The epithet “secondary metabolism” already familiar to plant physiologists was further promoted by the English microbial chemist J . D. Bu’Lock (1961). In his elegant review, Bu’Lock exquisitely states, Given the gencrally acceptable view that there are basic patterns of‘general metabolism, on which t h r variety of organic systems imposes relatively minor modifications, we can defino secondary mctabolism as having, by contrast, a restricted distribution (which is almost species specific) and 110 obvious function in general metabolism.

Whereas primary metabolites (sugars, amino acids, vitamins, nucleic acids, and polymers derived from them) are both essential and ubiquitous, making their presence universal among all organisms,” secondary metabolites” is the term used to collectively define those naturally occurring organic compounds that are unique to a small number of organisms. Even though secondary metabolites are nonessential to the organism that produces them, many of them have interesting biological activity (Table I). Since time immemorial, man used such metabolite extracts of plants as medicine to relieve pain and to cure diseases, as poisons in warfare and hunting, and as narcotics, hallucinogens, or stimulants. These interesting biological activities motivated curious chemists, who isolated and characterized the active princi-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

29

TABLE I SOME EXAMPLES OF SECONDARY METABOLITES Secondary metabolite Ephedrine Ricinine Salicin Strychnine Coniine Rotenone Morphine Tetrahydrocannabinol Cocaine Caffeine Ceraniol Linalool Cinnamaldeh yde Eugenol Diallyl sulfide

Use Respiratory ailments Castor oil purgative Antipyretic Poison Poison Fish poison and natural insecticide Narcotic Hallucinogen Stimulant Stimulant Perfumes Perfume Spice Spice Spice

Source Ephedra plant Castor oil Willow bark Strychnos plant Hemlock Opium Hashish; marijuana Cocoa Coffee Rose oil Lemon grass oil Cinnamon Cloves Garlic

ples from such plants. The structural complexity of secondary metabolites represents a great challenge to the synthetic chemists. Amino sugars, amino acids, quinones, coumarins, epoxides, alkaloids, glutarimides, glycosides, indoles, lactones, macrolides, naphthalenes, nucleosides, peptides, phenazines, polyacelylenes, polyenes, pyrroles, quinolines, and terpenoids are just a few examples of the diverse chemical structures represented by secondary metabolites. Unusual chemical linkages such as p-lactam rings, cyclic peptides, unsaturated bonds of polyacetylenes, polyenes, sterols, gibberellins, and the large rings of macrolides and ansamycins are peculiar to secondary metabolites. This diversity of a seemingly endless structural variety and complexity does not result from a multiplicity of basic building units but originates in a relatively small number of primary metabolites. Transformations of these precursors and their condensation with other moieties derived from central metabolic routes is responsible for the structural variety of secondary metabolites. Minor modifications of the number and the arrangement of carbon atoms of basic skeletons can yield a multiplicity of related chemical structures. Introduction of oxygen, nitrogen, chlorine, and s u l h r or changes in the oxidation level can further alter the hnctionality of the emerging metabolite. The study of secondary metabolism began in 1896 when Ernest Duchesne, a French medical student, discovered the low toxicity of extracts from Penicillium glaucum. These extracts probably contained penicillin, which was discovered in 1928 by Sir Alexander Fleming at St. Mary’s Hospital, London. However, it was not until World War I1 that research efforts to develop penicillin into a therapeutic agent were successful (Chain, 1980). H.

30

VEDPAL SINGH MALIK

W. Florey and Norman G. Heatley had worked with penicillin at Oxford. In 1941, England was undergoing air raids by the Nazi air force. Therefore, Florey and Heatley felt that penicillin work could be done better in the United States. They received financing from the Rockefeller Foundation to visit and enlist scientific collaboration of United States scientists. They contacted 0. E. May, the director of the Northern Regional Research Laboratory at Peoria, who received the following telegram from the United States Department of Agriculture headquarters: Thorn has introduced Heatley and Florey of Oxford, England, here to investigate pilot scale production of bacteriostatic material from Fleming’s penicillinm in connection with medical defense. Can you arrange irninrdiately for shallow pan srt-up to establish laboratory results. . . ?

0. E. May responded that the Northern Regional Research Laboratory was ready “to cooperate immediately.” Robert D. Coghill, Chief of the Fermentation Division, directed the penicillin project, which was terininated in 1945. Even though K. B. Raper, the mycologist on the project, isolated hundreds of strains of penicillin-producing fungi from soils collected by the Army Transport Command, the highest penicillin-yielding Penicilliurn chrysogenurri was isolated from a moldy cantaloupe obtained in a Peoria fruit market. During World War 11, industrial exploitation of the capability of hngi to produce penicillin received unprecedented attention. Commercial success of penicillin production not only laid the foundation for a prosperous billion-dollar fermentation industry but stimulated interest in the study of other microbial metabolites that might be of economic value (Rose, 1980). The Gascinating story of penicillin has been narrated in a superb manner by Chain and Raper and describes the efforts, good luck, and politics involved in commercializing the scientific observation that could otherwise have gone unnoticed for decades (Chain, 1980). During the early part of this century, Alsberg and Black 1912) reported the formation of an intriguing organic acid b y the cultures of Penicilliuin puberculun~They called it penicillic acid and noted its antiseptic action and moderate toxicity. More than a decade later Raistrick, working at the Nobel’s Explosives Company in Ardeer, Scotland, initiated investigations on the structures of fungal secondary metabolites. In 1929, Raistrick moved to the London School of Hygiene and Tropical Medicine, and he and his associates isolated more than 200 mold metabolites and determined the structure of penicillic acid. Since then the structures of numerous secondary metabolites have been determined and many such compounds are produced commercially. Thus, for example, almost 6000 antibiotics have been discovered and about 100 of them are the backbone of a multibillion-dollar, high-profit fermentation industry. Birkinshaw et al. (1936)showed that many secondary

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

31

metabolites are formed during the stationary growth phase. Over the past 40 years, biosynthesis of secondary metabolites has been studied mainly by tracer methods. Mutants blocked in metabolic pathways have been used in only a few instances to establish the entire metabolic route of complex products originating from the intermediates of primary metabolism. Brute-force mutagenesis has been widely used to develop microbial strains that are being used for commercial production of many products. Technologies that exploit the metabolic plasticity of microbes will play an increasing role in the near h t u r e and solve the shortages of some chemicals, solvents, fuels, and energy. Many other products that are of intricate chemical structure and that are hard to synthesize chemically may be rnicrobially produced without generating toxic waste (Whitaker, 1980). Versatile metabolic machinery of diverse microbes also may be employed to economically produce valuable compounds and their derivatives (antibiotics, alkaloids, human interferon, insulin, ethanol, long chain alcohols, acetic glycerol, ethylene oxide, acrylic acid) from renewable resources and thereby reduce pollution that is generated by an energy-consuming, expensive chemical synthesis. The application of modern methods of molecular genetics and recombinant DNA technology to the study of secondary metabolism could provide some useful solid information quickly. There exist limitless opportunities in the exploration of secondary metabolism, the study of which can now be speeded up since new methodology can circumvent the biochemical and genetic studies that hardly exist for the organisms that produce such metabolites. The purpose of this article is to give a general perspective of secondary metabolism and to provide a stimulus for investigators to think deeply about the scientific issues and other interesting aspects of the biology of the organisms that produce industrially important secondary metabolites.

II. Illegitimate Genome Sequences Even though secondary metabolism is not universal, the same secondary metabolite is produced by many organisms in widely separated taxonomic groups. Therefore, secondary metabolite production may have no relevance to the taxonomic classification of organisms. The occurrence of gibberellins in plants and Fusariuin and of ergot alkaloids in morning glory as well as Claviceps purpurea indicates that genetic capabilities for the synthesis of these metabolites are distributed among organisms belonging to different taxonomic groups. The occurrence of the P-lactam nucleus in wild-fire toxin produced by Pseudomonas tnbuci (Stewart, 1971) and in many p-lactam antibiotics produced by various Streptomyces, Nocardia, Penicillium, and Cephalosporiuin species (Aoki and Okuhara, 1980; Gorman and Huber, 1979) could be taken as another suggestive piece of evidence of illegitimate

32

VEDPAL SINGH MALIK

genome mixing and reassortrnent among various organisms. Production of chemically similar antibiotics by taxonomically remote species (Wagman and Weinstein, 1980) is further evidence for the same mechanisms of synthesis of secondary metabolites and for the existence of common pathways of secondary metabolism in different species. Similar cellular metabolites are generally, but not always, derived via similar biosynthetic sequences. The same, but sometimes different, taxonomic groups utilize the same metabolic pathway and may possess the same or similar enzymes and regulatory mechanisms to assemble similar types of chemical structures. Many secondary metabolites, including antibiotics produced by taxonomically unrelated organisms, contain the same subunits or substituents (Malik, 1980~).The genes for the subunits that are present in various secondary metabolites may be carried on transposable elements. Mixing and reassortment of these transposons resulting from illegitimate dissemination of genome sequences between organisms of various taxonomic groups might have conferred the ability to produce hybrid molecules with novel biological activity. For example, rubradirins (Fig. 1)consist of subunits that are present in three M e r e n t families of antibiotics as diverse as ansamycins (rubransarol), novobiocins (coumarin), and the everninomicins (dipicolinic acid) (Hoeksema et a ] . , 1979). P-Lactamases are widespread in enterobacteria (Richmond and Sykes 1973; Sawai et al., 1980),nocardias (Wallace et ul., 1978), streptomycetes, micromonosporas, and other nonstreptoinycetes actinomycetales (Schwartz and Schwartz, 1979). Of the other antibiotic-inactivating enzymes coded by eubacterial plasmids, those that acetylate chloramphenicol (Wright and Hopwood, 1977) and those that adenylate, acetylate, or phosphorylate aminoglycosides are present in streptomycetes (Davies et al., 1979). Location of the genes determining the structure and synthesis of antibioticinactivating enzynies on transposable elements could have favored their dissemination by illegitimate recombination among various microorganisms. Many of these antibiotic-inactivating enzymes are coded by chromosomal genes (Wright and Hopwood, 1977; Davies and Smith, 1978) in Streptoniyces. The modern methods of recombinant DNA technology can be used to study the similarity among enzymes of diverse origin and to determine if these chromosomally coded enzymes in the streptomycetes are a result of the insertion of the antibiotic-resistance-determining transposons into the chromosomes.

111. Enzymes of Secondary Metabolism The enzymes associated with secondary metabolites can be divided into four classes:

I

CONH,

Novobiocin

(Strepfomycesniueus)

FIG. 1. Similar subunit structures found in unrelated antibiotics.

34

VEDPAL SINGH MALIK

1. Those of primary metabolism that yield the precursors of secondary metabolism. 2. Those that are specific for the synthesis of the secondary metabolites and that convert the branch-point intermediates into final secondary products. 3. Those that provide energy or cofactors, or that insert various functional groups into the biosynthetic intermediates. 4. Those that further metabolize the produced metabolite. Even though the general outline of the biosynthetic pathways leading to various secondary metabolites is known, the details of the biosynthetic sequences have been elucidated only in a few cases. Mutants blocked at each step of the entire pathway are hardly available for any given metabolite, and little is known about the enzymology of the reactions involved. Indeed, the enzymes of secondary metabolism have proved difficult to purfi. This may, in part, be attributed to the fact that often secondary metabolism is at its peak of activity when general metabolism is slowing down, when cells are dying and undergoing proteolysis.

A. INDUCIBLEENZYMES Even though the reaction mechanisms involved in secondary biosynthesis are not essentially different from those of general metabolism, each secondary metabolite is made by a unique pathway. The formation of many secondary metabolites occurs by multistep processes catalyzed by multienzyme complexes that usually are produced only during a certain growth phase. Thus some of the antibiotic-synthesizing enzymes are induced during a short period at the end of the logarithmic growth and the onset of the stationary phase (Martin and Demain, 1978). They are also unstable and are in many cases inhibited by the products of the reaction they catalyze. As a result, most of the published curves that show the relationship between enzyme activity, growth, and the secondary metabolite production could be misleading because the enzyme preparation used for plotting such curves was not extensively purified to remove inhibitory enzymatic reaction products. Anhydrotetracycline hydratase of tetracycline-producing Streptoinyces aureofaciens uses NADPH and oxygen to hydrate anhydrotetracycline. The specific activity of this hydratase enzyme in the high-tetracycline-producing strain was one order of magnitude higher than the low producer. The activity of the enzyme increases rapidly around 24 hr of growth. Toward the end of growth, the enzyme activity diminishes. The addition of phosphate to the tetracycline-producing medium caused a decrease in specific activity of the hydratase enzyme; this decrease corresponded with decreased tetracycline production (Z. Hostalik, Czechoslovak Academy of Sciences, Prague).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

35

Streptomyces griseus contains an enzyme for the formation of dihydrostreptosyl streptidine-6-phosphate from enzymatically prepared dTDP-Ldihydrostreptose and chemically prepared streptidine phosphate. The enzyme has a molecular weight of 70,000 and is composed of two apparently identical subunits. The enzyme requires 10-12 mM Mg2+and is stabilized by streptidine. Chemically phosphorylated streptidine is the best acceptor, but streptidine and streptamine can also function as acceptors of dihydrostreptose. This enzyme may be used to make analogs of streptomycin where streptidine could be substituted by other aminocyclitol subunits in the enzymatic reaction under altered incubation conditions. During the course of streptomycin production in the S . griseus fermentation, the activity of this transferase enzyme appears at 1day, peaks at 2 days, and then declines. This parallels the activities of dTDP-L-dihydrostreptose synthetase and aminotransferase involved in the formation of streptidine (Kniep and Grisebach, 1980). In Streptontyces erythreus, propionate kinase is increased in stationary phase of growth (Raczynska et al., 1973). The acetyl-CoA and propionyl-CoA carboxylase activities in a macrolide producer reached a maximum at the onset of antibiotic production and decreased about threefold during idiophase. Conversely, 6-methylsalicylic acid synthetase is induced in the logarithmic growth phase and yields 6-methylsalicylic acid, a precursor of patulin, which accumulates in the idiophase. In Cephalospwium acremonium, the enzymes involved in the ring expansion and cyclization reach maximum specific activity 13 hr after the fungus has stopped growing, whereupon the enzymes are rapidly inactivated (Demain, 1980). Toward the end of the logarithmic growth phase and shortly before the onset of tyrocidine synthesis, the antibiotic-synthesizing enzymes and carrier proteins required for transporting the constituent amino acids of tyrocidine are induced. In the later stages of the stationary phase, insoluble enzyme preparations for tyrocidine biosynthesis were obtained from Bacillus brevis and were unstable and sedimented with the particulate fraction. Although the antibiotic production was enhanced in the later phase (Lee et al., 1975), a change of solubility of the tyrocidine-producing enzymes with the appearance of forespore in B . brevis suggests a relationship between antibiotic production and spore formation. Spore membrane-bound enzymes produce tyrocidine enclosed in forespore and induce cell transitioning into sporulation from the vegetative phase (Lee et al., 1975). B. COMPARTMENTALIZATION The efficiency of microbial synthesis is attributable to genetically controlled enzymatic levels and special enzyme properties. Intracellular compartment-

36

VEDPAL SINGH MALIK

alization of enzymes, substrates, and cofactors makes the biosynthetic processes highly efficient. Multienzyme complexes with catalytic subunits arranged to catalyze successive steps of synthesis are known to be involved in the biosynthesis of secondary metabolites. The catalytic efficiency of these complexes is probably higher in the intact cell than in cell-free extracts because the intracellular intermediates are largely enzyme-bound during the biosynthetic process. Toxic enzyme-bound intermediates may not inhibit cellular functions because they are never free in the cytoplasm. In the cellfree extracts, on the other hand, this delicate order is disturbed with the ensuing dilution of the desired enzymatic activities.

C . SPECIFICITY Enzymes that carry out primary metabolic processes have evolved with strict substrate specificity because their product is essential to the growth of the organism. On the other hand, enzymes involved in the biosynthesis of secondary metabolites have not always been selected for rigid specificity because their products are not usually essential for the growth of the producing organism. Unnatural compounds that are formed as a result of the action of these loosely specific enzymes may serve as substrates for the synthesis of new structures. Because of the loose substrate specificity, secondary metabolites are produced as families of closely related molecules with minor modifications of a basic structure. For example, there are about a dozen natural penicillins, thienamycins, and olivanic acids, 3 neomycins, 20 rifamycins, 4 tyrocidines, 5 mitomycins, 10 bacitracins, 10 polymyxins, 20 actinomycins, 4 levorins, 4 polifungins, 13 bleomycins, and numerous sporedesmins. One Mi~romonospm-aproduces about half a dozen aminocyclitol antibiotics (Berdy, 1974). Streptomyces tenebrarius produces a complex of aminocyclitol compounds called nebramycin, with a dozen known chemical structures. The ratio of the components in the fermentation mixture varies greatly and depends on growth conditions. Several components have been characterized as apramycin, kanamycin B, and tobramycin (Stark et al., 1980). Caerulomycin is another major solvent-extractable antibiotic coproduced with nebramycin complex (Funk and Divekar, 1959). The number of antibiotic-like molecules, of which only a few are biologically active, is very large and is the result of a variety of biochemical and stereochemical transformations that occur during antibiotic synthesis. For instance, 72 tetracycline-type compounds can originate from a hypothetical nonaketide intermediate. Twenty-seven of them have been isolated (Hostalek et nl., 1979). In the synthesis of penicillins, the final acylation step is not specific. Many acids similar to phenylacetic acid can be utilized to synthesize penicillin G

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

37

analogs. In the biosynthesis of some oligopeptides and polyketides, intermediates are formed by nonspecific reactions as any one of several alternative amino acids can be incorporated in the middle of the peptide antibiotics and none limits overall synthesis (Queener et al., 1978). The synthetases involved in the biosynthesis of bacitracin, polymyxin, and gramicidin can also incorporate amino acid analogs into the final molecules. Very frequently circular peptides are produced as a mixture of a variety of compounds thus making their purification discouragingly tedious. Synthetases are nonspecific to such a degree that they will accept and incorporate one of several amino acids and yield a mixture resulting from substitution of various amino acids in the final molecule. Enniatin, the cyclohexadepsipeptide antibiotic, has alternating residues of D-hydroxyisovderic acid and N-methyl amino acids, forming an 18membered ring. The enniatins A, B, and C contain the N-methyl derivatives of L-isoleucine, L-valine, and L-leucine, respectively. All three depsipeptide enniatins (A, B, and C) are synthesized by the same soluble multienzyme complex. The enzyme has been purified to a high degree from Fusarium and has a molecular weight of 250,000. Synthesis of specific types of enniatins is thus dictated by the intracellular concentrations of various amino acids that are subsequently assembled into different depsipeptides. Omission of S -adenosylmethionine results in the production of nonmethylated enniatins. The rate of formation of nonmethylated enniatins is only 15% of that of enniatins that require S-adenosylmethionine as a methyl donor. In biosynthetic schemes of certain secondary metabolites, even the exact order of reactions may not be important. Several parallel pathways may convert a given intermediate to the same end product. For example, several parallel routes probably convert lanosterol to ergosterol. An enzyme system of this kind can be used to carry out regiospecific one- or two-step transformations of unnatural sterols, which is of economic importance for biological transformation not only of sterols, but also of other molecules. If an enzyme that participates in the biosynthetic sequence as a group modifier is missing, then further enzymatic modifications still occur. For example, if either methionine or methylating enzyme is depleted from the cell, the demethylated tetracyclines are produced. Even though methylation is one of the earlier steps in tetracycline biosynthesis, further modifications of the demethylated precursor by succeeding enzymes proceed in the usual manner. The dehydrogenation step in the synthesis of many secondary metabolites is of economic significance. In the same fermentation, saturated and unsaturated compounds may occur as mixtures and their occurrence depends on fermentation conditions. Cholesterol and dihydrocholesterol, ergosterol and dihydroergosterol, gibberellins and dihydrogibberellins, fusaric acid and di-

38

VEDPAL SINGH MALIK

hydrofusaric acid, streptomycin and dihydrostreptomycin, spectinomycin and dihydrospectinomycin occur in varying ratios in the same fermentation. Dehydrogenation may be a reversible step, and understanding of the regulatory mechanism that controls this step may be used to direct the fermentation to minimize or to avoid undesirable species of metabolite. As a result of the loose specificity of enzymes involved in secondary metabolism, cellular synthetic machinery has been used to incorporate analogs of precursors for generating altered molecules (Malik, 197913).

IV. Controlling Effect of the Environment

A. NUTRITION Although good growth may occur in many media, secondary metabolites may only be produced in a specific medium. Thus Penicillium cyclopium forms penicillic acid on Raulin medium but not on Czapek-Dox medium (Bentley and Keil, 1962). Sometimes a given organism may produce one metabolite on one medium and a totally different one on another medium. For example, Penicillium griseofuluum produces griseofulvin on CzapekDox medium and fulvic acid on Raulin medium (Oxford et al., 1935). Variation in the chemical composition of the medium and its relationship to yields and type of secondary metabolites is well known. Development of a medium that produces high yields of a desired secondary metabolite is still empirical to a considerable degree (Demain, 1972, 1973). Distribution of precursors into various branch pathways may vary and may depend on the growth environment and the genotype of the organism. The flow of precursors can be increased in the desired direction by mutation or medium manipulation. Brewer and Frazier substituted gum (dextrin) for glucose to produce amphotericin B in preference to amphotericin A. The presence or absence of certain ions or of carbon and nitrogen sources can inhibit, activate, induce, and derepress certain enzymes, perturbing the normal channeling of key intermediates that support balanced growth (Weinberg, 1978). B. TRACEMETALS Depression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli by growth in iron-deficient medium has been reported by McCray and Herrmann (1976). Iron-deficient medium is the key to the commercial production of citric acid. Trace metals play a remarkable role in secondary metabolism and may be involved in enzyme-coenzyme combinations. For example, a zinc-deficient culture of Rhizopus nigricans accumulates large amounts of fumaric acid. However, in the presence of small amounts of zinc, primary metabolism

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

39

becomes predominant and no fumaric acid is produced (Foster and Waksman, 1939). In 1955, Ehrensvard noted that the major metabolite is 6-methylsalicylic acid when Penicilliuin urticae is grown on Czapek-Dox medium containing lo-* N Zn2+. However, with N Zn2+ in the same medium, the 6-methylsalicylic acid is not formed, but large amounts of gentisic alcohol, toluguinol, and patulin accumulate instead. The effect of the level of iron on the ratio of patulin to gentisyl alcohol production by a Penicillium was reported in 1947 by Brack. Multiple antibiotic moieties or totally different biologically active molecules can be produced in low yields during fermentation. In such cases, the use of various media results in diEerent antibiotic ratios. It becomes challenging to purify a highly active component that is produced in tiny amounts in a complex medium. Godfrey and Price (1972) identified many different components in a coumermycin fermentation. It was a stroke of good fortune to find that traces of cobalt ion directed the biosynthesis exclusively to couinermycin A , . Producing organisms may require vitamin B,2 in order to methylate antibiotic precursors and need traces of cobalt to ensure enough vitamin B,, to complete that sequence. The metabolite resulting from a fermentation in which biosynthesis has been directed mainly to the production of one component is easily isolated.

C. HYDROGEN IONCONCENTRATION p H has profound effects on both primary and secondary metabolism (Foster, 1949). For example, the best yields of itaconic acid by shake cultures of Aspergillus terreus are obtained when the organism is grown at a constant pH of 1.8. Any increase in the initial pH leads to increased cell mass but decreased itaconic acid. p H 2.3 is optimal in surface cultures, whereas below pH 2.2 and above p H 2.4 no itaconic acid is produced (Lockwood and Nelson, 1946). For maximum yield of kojic and citric acids, Aspergillus niger should be inoculated into a medium with pH 2 . 5 3 . 5 . A higher initial p H shifts the metabolism toward the formation of oxalic and gluconic acids.

D. TEMPERATURE AND AERATION Every fermentation process has an optimum temperature and aeration rate.

V. Genetics of Secondary Metabolism The rationality inherent in the techniques of modern genetics may disentangle many intellections about secondary metabolism. Genetics studies of secondary metabolites have been repeatedly

40

VEDPAL SINGH MALIK

scrutinized (Queener et al., 1978; Hopwood and Merrick, 1977; Malik, 1979a; AliKhanian and Danilenko, 1979), but experiments dealing with the mapping of genes determining defined enzymatic reactions are scarce. In fact, all biosynthetic steps, using blocked mutants and enzymes, have not been defined for any single secondary metabolite. Systematic and precise study of the biochemical genetics and regulatory systems of secondary metabolite-producing microbes are progressing rather slowly. Therefore, it is hard to formulate any sound hypothesis regarding regulation of secondary metabolism. Because of the economic importance of antibiotics, many efforts have been made to obtain antibiotic-overproducing strains by genetic recombination (AliKhanian and Danilenko, 1979; Malik, 1979a; Cape, 1979; Hopwood, 1978; Hopwood and Chater, 1980), but the results thus far have not been very encouraging. Synthesis of a secondary metabolite is a polygenic characteristic (Malik, 1979a). Vanek et al. (1971) estimated that more than 200 genes are involved in the synthesis of chlortetracycline. The genes directly governing synthesis of antibiotics from basic building subunits are involved, but those responsible for the synthesis of their precursors, coenzymes, cofactors, energy metabolism, transport mechanisms, cell permeability, architecture, and resistance to the produced antibiotic also affect final antibiotic yield. Various genetic methods developed with Streptomyces coelicolvr have been used by many investigators to study the genetics of various other streptomycetes (Hopwood and Merrick, 1977). AliKhanian and co-workers performed many experiments to unravel the genetic control of oxytetracycline biosynthesis in Streptomyces rim.osus, and they discovered that an auxotrophic mutation decreased or abolished antibiotic activity in S. rimosus. Deleterious and pleiotropic effects of auxotrophy on antibiotic production make it hard to map genes involved in antibiotic synthesis. Various investigators have used multiply marked auxotrophic strains in curing experiments to suggest involvement of plasmids in antibiotic synthesis. These results should be confirmed using a prototrophic, high-producing ancestor. Mutations that map at three different locations on the S. coelicolor chromosome are pleiotropic, because they nullify the production of methylenoinycin, actinorhodin, and aerial mycelium (Merrick, 1976). It would be useful to have a parallel attack on a few antibiotic-producing systems. The study of chlorainphenicol production by Streptomyces uenezuelae, of cephamycin production by S. griseus, of actinorhodin production by S . coelicolw, of tetracycline production by S. rimosus, and of j3-lactam production by several microbes have already been pursued to a level where meaningful experiments can now be designed to obtain answers to specific questions. Recombination occurs in many other secondary metaboliteproducing microbes (Hopwood and Merrick, 1977) but cannot as such be

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

41

exploited to study metabolic regulation, because no practical means of performing a genetic complementation test exists in secondary metaboliteproducing organisms. Recombinant DNA technology will now facilitate the construction of merodiploids, allowing a complementation test and operon fusions. This will aid in the dissection and manipulation of regulatory mechanisms for commercial production of these metabolites (Malik, 1980b).

A. CHROMOSOMAL MAPPING Genetic material for primary pathways is organized into regulatory units, and genes of many pathways are clustered in one segment of the genome. A gene cluster that is involved in a single pathway is called an “operon.” Organization of genes in operon-like structures is efficient but not essential for coordinated regulation of a metabolic route. Genes of many pathways do not form single gene clusters (operons) but are scattered in various segments of the chromosome. When genes of a metabolic pathways form several gene clusters (minioperons) located at various sites on the chromosone, they still can have tight regulatory interdependence. All the minioperons that are coordinately regulated have been called “regulons” (Goldberger, 1979). By using complementation and cosynthesis, about nine structural genes involved in oxytetracycline synthesis have been located on the chromosome in two clusters (AliKhanian and Danilenko, 1979). The fact that about 200 genes may be involved in tetracycline biosynthesis makes it clear that a great deal remains to be done to completely understand the genetics of oxytetracycline production. In the meantime, by mutagenesis, the yield of tetracycline has been increased to more than 25 gmfliter. Another report of the chromosomal location of genes involved in secondary biosynthesis has been provided by Wright and Hopwood (1977). A series of 76 point mutations, causing blocks in actinorhodin production, map in a cluster on the S. coelicolor chromosome (Rudd and Hopwood, 1979), suggesting that most genes involved in actinorhodin biosynthesis are closely linked. Mutants blocked in actinorhodin biosynthesis fell into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors, and cosynthesis. Actinorhodin (Brockmann et al., 1966), kalafungin (Hoeksema and Krueger, 1976), nanaomycins (Omura et al., 1976), griseusins (Tsuji et al., 1976), granaticin, and the naphthocyclinones belong to the isochromane quinone class of antibiotics (Zeeck et al., 1974). Study of the genetics of actinorhodin biosynthesis by S. coelicolor could be relevant to the understanding of the biochemistry and genetics of the polyketides. Actinorhodin is a pH indicator. It is blue and very soluble in polar solvents above p H 7. Below pH 7, it is red and sparingly soluble. On certain media, this acid-base

42

VEDPAL SINGH MALIK

indicator pigment accumulates intracellularly in the red, acidic, waterinsoluble form (protoactinorhodin). The red protoactinorhodin is converted to the blue, water-soluble form by inverting the culture agar plates over ammonia solution in a petri dish lid. Mutants blocked in actinorhodin do not respond to ammonia fumes and do not change color from red to blue (Rudd and Hopwood, 1979). This simple assay, based on visual detection of colony color, makes genetics of actinorhodin rather easy. Another attractive feature of actinorhodin is the fact that this pigment is produced by S . coelicolur, whose genetics and molecular biology are by far the most advanced among all streptomycetes (Hopwood, 1980b; Chater, 1980). The genes that control biosynthesis of actinorhodin and another red pigment are closely linked in a single chromosomal cluster (Rudd and Hopwood, 1980).

B. EXTRACHROMOSOMAL ELEMENTS Although the knowledge of the genetic control of secondary metabolism is rather fragmentary, there are indications that plasmids may play a role in it. Because secondary metabolism is not essential for the survival of the producing organism and plasmids usually code for such secondary functions, it is possible that certain plasmid DNA sequences play a role in the synthesis of secondary metabolites (Kalakoutskii and Agre, 1976). This role may be direct. For example, structural genes for methylenomycin A biosynthesis in S . coelicolur appear to be plasmid borne (Hopwood, 1980). It is possible that the genes that code for the metabolic steps of secondary metabolite production are clustered in operons and are located on plasmids. There are four classes of evidence that suggest involvement of plasmids in the synthesis of antibiotics. They are considered in the following sections. 1, Natural Genome lnstability

Many researchers working with secondary metabolites have observed that metabolite production is frequently lost. Many such examples, including observation of unstable colony pleomorphism, have been reported (Malik, 1979a; Nakatsukasa and Mabe, 1978). The polygenic character of secondary metabolism may be partly responsible for the frequency of genetic degeneration of industrial strains. Another cause of variation in prokaryotes is associated with the extrachromosomal genetic elements known as plasmids. Location of genes on a plasmid ensures that (depending on the plasmid status of the cell) the cell gains or loses a block of genetic material. These additional novel genes could be of survival advantage to that cell in an existing environment or in adjustment to a new one. One example of a genetic element involved in genome instability is the

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

43

element Tyl of yeast (Greer and Fink, 1979; Roeder et al., 1980; Scherer and Davis, 1980). A laboratory strain of Saccharoinyces cwevisiae contains 30 copies of Tyl per cell. It accounts for over 1%of the whole yeast genome. It is probably a virus of about 5 kilobases in length. It has duplications on the end that are called delta. The whole element, including duplications at the end, is transcribed, and the corresponding RNA accounts for 10% of the total RNA of yeast. Thus, Tyl constitutes a large fraction of the genome and produces a large fraction of RNA in the yeast. The function of Tyl is not known. It has no known phenotype. However, it can be tagged and then mapped by fusion to other genes of known phenotype (Roeder and Fink, 1980). Cameron et al. (1979) fused His3 gene in the middle of Tyl by in uitro manipulation and transformed the His3 -containing Tyl element into yeast, which integrated it into the chromosome by homologous recombination at many different locations. These integrated sequences are scattered over the whole yeast genome and recombine if they are located on different chromosomes; this results in scrambling of the yeast genome. The genetic element Tyl is probably responsible for most of the reciprocal translocation occurring in yeast (Chaleff and Fink, 1980). Gene conversion has been demonstrated by Scherer and Davis (1980) using the repetitive DNA of Tyl. These investigators fused a promoterless nonfunctional His3 gene into one Tyl element and another nonfunctional His3 gene with a deletion in the middle into another Tyl element. Both of these genes were inserted into the chromosomes of yeast to determine what events might occur to reestablish a functional His3 gene. A classical revertant should not restore function because both His3 genes are nonfunctional because of deletions. Some major rearrangement of the genome would be required for reestablishing function. In fact, these deletions were gene converted across chromosomal arms into a functional His3. These surprising results suggest that one gene sequence, without any change in its own sequence, can gene convert the other. Chromosomes with similar sequences can communicate with one another. One can even insert large pieces of DNA in the middle of His3. Davis inserted a galactose gene cluster into His3. That cluster was gene converted out to restore functional His3, suggesting that all that is required for gene conversion is flanking the DNA sequence homology at both ends of the sequence. Since repetitive DNA is very prevalent among microbes (Klein and Welch, 1980), this kind of phenomenon could rapidly rearrange genomes of organisms of industrial importance. Transposable element Tyl also affects gene expression. It does not affect gene expression if inserted in the middle or beside the gene. It only turns genes on if inserted at a distance. The frequency of these events are lo4. They act recessive and are cold sensitive.

44

VEDPAL SINCH MALIK

Other secondary metabolite-producing organisms like streptomycetes and fungi may have repetitive DNA sequences like Tyl. Therefore, they could also recombine and produce genome instability as a result of inversions, translocations, and deletions. These elements can also communicate with one another without being translocated or excised. This could result in gene cwnversions similar to those in yeast.

2 . Curing The naturally occurring instability of antibiotic production is further enhanced if a culture is treated with DNA chelating agents such as ethidium bromide or acridine orange. Antibiotic-negative variants occur at very high frequency (1to 10%or more) among the populations that have been exposed to UV, high temperature, novobiocin, or rubradirin. Clones that have been regenerated from protoplasts also yield a large proportion of antibiotic nonproducers. A number of plasmicl curing agents have been tried, and only treatment with novobiocin resulted in loss of ability of Streptomyces refuineus var. thermotolerans to produce anthramycin. The anthramycin nonproducers of S . refuineus were less resistant to anthramycin, had lost their typical leathery colonial appearance on plates, and produced a light-activated pink pigment not normally produced by the anthraniycin-producing strains. Similar plasmid DNA was present in both the anthramycin producer and the nonproducer. Novobiocin may cause deletions resulting in pleiotropic effects, because no reversion back to antibiotic production occurred even after repeated transfers (J. Stefan Rokem and Laurence H. Hurley, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506). The ability of Streptomyces kasugaensis to produce aureothricin, thiolutin, and kasugamycin is eliminated by treatment with curing agents. Two plasmids (6.8 and 15 megadaltons) have been isolated from S . kasugaensis, but further evidence is required to establish the role of these plasmids in antibiotic production. Curing studies with Streptomyces alboniger suggest that specific functions required for aerial mycelial formation, including pamamycin production, may be coded by extrachromosomal elements (Pogell, 1975; Redhaw et al., 1979). The curing action of acriflavine suggests the possible involvement of extrachromosomal elements in controlling aerial mycelia, pigment, and avermectin production in Streptomyces avermitilis. Other antibiotics that have been reported to be plasmid controlled are chloramphenicol (Akagawa et al., 1975), holomycin (Kirby, 1978), kasugamycin, aureothricin, methylenomycin (Bibb et al., 1980a), turimycin, chlortetracycline (Boronin and Sadovnikova, 1972) streptomycin (Shaw and Piwowarski, 1977), actinomycin (Ochi and Katz, 1978), neomycin, spiramy-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

45

cin (Omura et al., 1979), kanamycin (Chang et al., 1980; Hotta et al., 1977), and beromycins (Blumauerova et al., 1980b). Akagawa et al. (1975) have reported that upon acridine treatment of chloramphenicol-producing S. venezuelae, many nonproducer clones were obtained among the progeny. The fact of loss of chloramphenicol production at high frequency and further genetic analysis were used to suggest involvement of a plasmid in chloramphenicol biosynthesis (Akagawa et al., 1975). If chloramphenicol nonproduction in S. venezuelae is indeed attributable to permanent loss of a plasmid, then the cured strain should be incapable of reverting to chloramphenicol production. However, revertants of the strain claimed to have been cured of chloramphenicol production by Akagawa et al. (1975) have been selected (Malik, 1980~).These revertants simultaneously produce chloramphenicol (50 mg/ml) and are resistant to chloramphenicol(1OO mg/ml). This shows that the presumptive cured strain has not lost the genetic potential to synthesize chloramphenicol. Therefore, alternative interpretations have to be considered to explain this loss of the synthetic capacity in the pseudo-cured strain of Akagawa et al. (1975). S. venezuelae (VM3: his-leu-ade-cpp-) does not grow vigorously as some other chloramphenicol-producing Streptomyces (Malik, 1979b). The poor growth of S. venezuelae (VM,: his-leu-ade-cpp-) may be attributable to the presence of several auxotrophic markers and could influence the yield of chloramphenicol. A study of the effect of reversion to prototrophy on chloramphenicol production and growth might be of interest. Antibiotic production by Pseudomonas reptilivora as a result of phage conversion has also been reported (Martinez-Molina and Olivares, 1979). Nakano et al. (1980a) subjected Streptomyces lavendulae to curing agents and obtained unstable pleiotropic mutations at high frequency. Most of the auxotrophs obtained were arginine auxotrophs, required argininosuccinate for growth and either did not produce any P-Iactamase or produced a low level of p-lactamase. Arginine auxotrophs also failed to produce any aerial mycelia, formed small colonies, and showed increased sensitivity to benzylpenicillin. Nakano et al. (1980b) found only arginine auxotrophs at a frequency of 12%among plasmid-carrying S. kasugaensis cells that had been treated with acriflavine. Arginine auxotrophs did not contain any plasmid. Ethidium bromide treatment produced revertant prototrophs at a frequency of 1 0 -to ~ lo-'' and these revertants were shown to regain the plasmid. Arginine auxotrophs spontaneously reverted to prototrophs at a very low frequency. These authors suggest that an unstable genetic element regulating secondary metabolism inactivated argininosuccinate synthetase gene by transposition or insertion. Similar phenomena have also been reported in other

46

VEDPAL SINGH MALIK

Streptomyces (Pogell, 1975; Redshaw et al., 1979; Sermonti et al., 1978, 1980). Genes involved in chloramphenicol resistance, argininosuccinate synthetase, chromosome transfer, and aerial mycelium formation may be carried on a transposon SCTn,, which has two high transposition specifity regions on the S. coelicolor chromosome, one between cysA and metA and the other at the right of urgA (Sermonti et al., 1980). As in other bacteria, the chloramphenicol resistance on SCTn, is not attributable to chloramphenicol acetyltransferase, but the transposon may code for permeability alterations. This type of evidence for involvement of plasmids in antibiotic synthesis is only suggestive and not definitive. Other causes of genome instability in many organisms are well known (Malik, 1979a, 1980~).Tandem genome duplications, chromosomal rearrangement due to inversions, and insertions of DNA sequence all lead to instability. Other mechanisms analogous to control of phage variation in Salmonella or activation of mating type in Saccharomyces cerevisiae may be responsible for genomic instability in Streptomyces. Such mechanisms could be involved in regulation and synthesis of antibiotics and other secondary metabolites. The mechanism of DNA packaging in the Streptomyces spores could be analogous to DNA packaging in bacteriophage T4, resulting in circularly permuted genomes with tandem duplications. Certain agents (e.g., ethidium bromide) could induce chromosomal rearrangements, whereas others simply may select naturally occurring variants with spontaneous genome rearrangements. 3. Extrachromosomal D N A

A good system of genetic analysis can yield information suggesting involvement of plasmids in the biosynthesis and regulation of a secondary metabolite. Recombination frequency showing infectious transfer and no linkage to chromosomal markers was used to conclude that genes that determine chloramphenicol and methylenomycin biosynthesis are plasmid borne (Hopwood et d., 1980b). However, the demonstration of the presence of plasmid DNA in the antibiotic-producing strains and the absence of plasmid DNA in the antibiotic-negative strains would provide more evidence of involvement of plasmids in antibiotic production. Such correlations have not yet been demonstrated. Rearrangement of plasmid or insertion of plasmid into the chromosome with accompanying secondary metabolite production is another piece of evidence that could suggest plasmid involvement. Transformation of antibiotic production by the isolated plasmid DNA into an antibiotic-negative strain would be the best evidence, but such solid data have not yet been obtained for any antibiotic. The plasmid pUC3 was isolated from S. uenezuelae strain 3022a, which

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

47

produces chloramphenicol. The plasmid was digested with the restriction endonuclease BamHI into four fragments with approximate sizes of 19, 5.7, 4.1, and 2.7 kilobase pairs. These fragments were cloned into Escherichia coli HBlOl using the plasmid pBR322 (having a single BamHI site) as a vector. None of the constructed E. coli clones produced chlorainphenicol in detectable amounts not did any of the clones express resistance to the 12 antibiotics tested. Marahiel (personal communication) has isolated a plasmid DNA from the cells of Bacillus brevis ATCC 9999 (gramicidin S producer) and of a gramicidin S-negative mutant. All of the plasmid DNA in the parent culture is associated with the chromosome and membrane, whereas the same plasmid is free in the cytoplasm of the mutant that does not produce gramicidin S. Interaction of plasmid with the membranes and chromosome may regulate genes involved in gramicidin S biosynthesis. The plasmid has a molecular weight of 41.2 x lo6 and does not seem to carry any resistance to antibiotics like lincomycin, novobiocin, kanamycin, or streptomycin. Microcins are low-molecular-weight (less than 1000) antibiotics produced by Gram-negative bacteria. They inhibit the growth of a wide range of bacterial genera by specific inhibition of methionyl-tRNA synthetase. Microcins also inhibit methionine synthesis because they act as competitive inhibitors of homoserine-0-transsuccinylase, an early enzyme involved in the biosynthesis of L-methionine. Plasmid codes for the synthesis of the microcin and resistance of the host cell to its action (Diaz and Clowes, 1980). I n no case has the antibiotic-synthesizing ability been transformed into a nonproducing Streptomyces by using purified plasmid DNA. Isolation of plasmids from macrolide-producing Streptomyces ( S . albogriseolus, S . griseoflavus, S. reticuli, and several others) was reported by H. Schrempf (Wirzburg, W. Germany). Cured variants of S . reticuli that have lost antibiotic resistance, antibiotic production, sporulation, and/or melanin production either have no plasmid DNA or contain plasmids with deletions and/or insertions. Expression of the tyrosinase gene was correlated with the presence of a 3-million-dalton plasmid fragment, which is lost in melanin-negative strains. However, only the transformation of melanin-negative strains to melanin producers by purified plasmid will provide the firm evidence that plasmid, indeed, directs melanin synthesis in S . reticuli. In some variants of S . reticuli, certain DNA sequences are amplified, and extensive reorganization of chromosomal sequences occurs. Similar observation of extensive genome instability and rearrangements was made in the neom ycin-producing Streptomyces faradiae. Strains of S . faradiae that have been cured of neomycin production contain somewhat different plasmids (Davis, 1980). One mutant (H3) that is resistant to high concentrations of neomycin yields five times more plasmid DNA and produces more phos-

48

VEDPAL SINGH MALIK

photransferase and neomycin . Restriction enzyme analysis of plasmid DNA korn H3 strains showed plasmid rearrangement. Parent plasmid was 52.1 megadaltons, but mutant H 3 plasmid was 56.4 megadaltons and had a 4-megadalton insertion near the two o'clock region. In other strains of S. furadiae that have different levels of resistance to neornycin, rearrangement occurs in the same region as in the parent plasmid. However, purified S. furucliae plasmid DNA did not hybridize with the purified genes of neornycin-3-phosphotransferase and neomycin-3-acetyltransferase that have been cloned in Hopwoods laboratory from the same S. furudiue strain. The coincidence of relationship between plasmid DNA, neomycin resistance, and neomycin production should be further examined by carrying out transformation of the neomycin-nonproducing streptomycetes with plasmid DNA.

c. GENETICENGINEERING OF SECONDARY METABOLITES Availability of host vector systems and several cloned genes provides probes for hybridization and selection, thus making molecular biology of these industrially important organisms amenable to further exploration. This knowledge is ripe for harvest-to achieve desired results in the fermentation industry. The new recombinant DNA methodology can be used to fuse genes and to construct merodiploids for performing a genetic complementation test yielding valuable information about cellular regulatory mechanisms. Several vectors can now be used to clone genes in streptomycetes. Fragments of S . coelicolor genome carrying methylenomycin resistance and various prototrophic alleles have already been cloned using SCPB* as a vector and S. coelicolor as a host (Malik, l980b). Such clones are highly unstable but can be stabilized if a fragment of SCP2* that carries a specific plasmid segregation fiinction is included in the recombinant plasmid. Hopwood arid associates cloned antibiotic resistance genes from S. farudiae and Streptomyces azureus into Streptomyces lividans. The S . lividans plasmid SLP1.2 was used as a vector to clone the neomycin-3phosphotransferase gene and the neomycin-3-acetyltransferase gene from S. farurliat.into S. livirlans. Using the same vector, thiostrepton resistance and erythromycin resistance from S . amreus and S . erythreus have also been cloned in S. liuidans. These antibiotic resistance markers can now be inserted into other vectors or combined together to build vectors with multiple resistances to generate a gene-transporting vehicle analogous to E . coli cloning vector pBR322. Many organisms, such as Drosophila, corn, and yeast (Stinchcomb et al., 1980), contain autonomously replicating sequences dispersed throughout their genome. Similarly, vectors can now be built totally out of chromosomal

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

49

sequences of industrial organisms; these vectors will replicate autonomously or integrate into the host chromosome. Such vectors yield very high transformation rates (Struhl et al., 1979a). Some vectors are unstable, but DNA can be inserted to enhance stability. For example, centromere sequences make yeast vectors relatively stable. The temperate actinophage C31 has been developed as a suitable Streptomyces cloning vector (Chater, 1980). Like all known Streptomyces temperate phages, this actinophage resembles E . coli phage lambda in size and morphology and contains sticky ends. A 20-gene linkage map has been constructed by using temperature-sensitive plaque morphology and host range mutants. Heteroduplex analysis, denaturation, and restriction enzyme digestion have been used to align the linkage niap with the physical map of the linear 39-kilobase D N A of C31. Repressor gene ( C )is located in the middle of the genome and many genes for essential functions are located to the left of the C gene. Actinophages that are resistant to chelating agents have deletions that are in the center of the genome and reniove repressor gene or that are in the rightmost 25% of the molecule and remove phage attachment site to the chromosome. Partial Eco RI digests of E. coli plasmid pBR322 and actinophage deletion mutant C31Cts23 were ligated and transfected into S. lividans. Transfectants were selected by hybridizing clones to radioactive pBR322 DNA. The resulting C32-pBR322 recombinant molecule replicates as a phage in Streptomyces and as a stable plasmid in E . coli. Further modifications have produced a vector that has single restriction sites for BamHI and PStI and can be used to clone up to 7 Rb of foreign DNA. A C31-pBR 322 hybrid is restricted by S. albus P and does not form plaques on R-m+ S. albus G mutants if part of pBR322 is inserted in C31. This fact may be used to select deletion mutants. Ampicillin and tetracycline resistance genes of pBR322 may be expressed in Streptomyces if inserted in such a manner that they are transcribed from an actinophage promoter. Chloramphenicol acetyltransferase gene from E. coli has also been cloned in S. lividans using S . lividans plasmid SLP1.2 as a vector. This enteric bacterial gene is expressed in S. lividans and its transcription initiates from the promoter of either Streptomyces vector or E . coli gene controlling elements are being recognized by streptomycetes transcription machinery. These cloned antibiotic resistance genes provide a good selective marker for cloning D N A sequences in streptomyces. The convenient assay of chloraniphenicol acetyltransferase and P-lactamase can be further used to understand the molecular biology of Streptomyces promoters. In this way, systematic study of the molecular biology of Streptomyces may thus be exploited to generate hybrid antibiotics, to introduce useful genes for utilization of inexpensive carbon sources (e.g., cellulose) and for production of valuable human metabolites (e.g., insulin, interferon). Multicopy vectors with highly efficient constitu-

50

VEDPAL SINGH MALIK

tive promoters should soon be available to amplify and to express genes of interest in Streptomyces, making them very attractive for the production of valuable inetabolites. Certain thermophilic Streptomyces that grow in a very high cell mass on cheap carbon sources are another promising host for producing valuable metabolites (e.g., amino acids, organic acids, vitamins, enzymes, antibiotics, coloring pigments) at a reduced cost. Cell-free biosynthesis of antibiotics and other secondary inetabolites is yielding knowledge of their biosynthetic processes and regulation as well. Kleinkauf and co-workers (Technische Universitat, Berlin) have purified many multienzyme complexes from various Bacillus species; these complexes are involved in the synthesis of gramicidin S, tyrocidine, bacitracin, polymyxin, and many other peptide antibiotics. The purified enzymes can be used to make radioactive antibodies that may be used to develop radioimmunoassays useful in cloning genes that determine the synthesis of enzymes involved in antibiotic production. Enzymology of chloramphenicol biosynthesis in S. venexuelae unravels some very interesting phenomena (Malik, 1979b). Chloramphenicol is derived from chorismate, and the branching enzyme arylamine synthetase converts chorismate to p-aminophenylalanine. This enzyme is not presept in cultures unable to produce chloramphenicol and is repressed in producing cultures by addition of chloramphenicol. Arylamine synthetase has a subunit with aminotransferase activity. This aminotransferase subunit is multispecific and may be shared by anthranilate synthetase, p-aminobenzoic acid synthetase, and arylamine synthetase. If the same aminotransferase subunit is, indeed, common to the branching enzymes involved in biosynthesis of tryptophan, p-aminobenzoic acid, and chloramphenicol, then study of the regulation of the synthesis of this multispecific aminotransferase subunit may yield interesting information. Nucleotide sequences for this subunit may be isolated by utilizing cloned anthranilate synthetase gene of E . coli, Bacillus subtilis, or yeast as a hybridization probe. DNA sequencing could reveal the mystery and advance our knowledge of the regulatory biology of streptomycetes, exposing the structures of attenuators, leaders, operators, promoters, and other nucleotide sequences involving regulatory elements. Judicious manipulation of these sequences could result in a high level of gene expression that would increase yields of desired metabolites, ending up with a cheap cost of production. Several streptomycetes that produce novel /3-lactam antibiotics (thienamycin, cephamycin, nocardicin) have been isolated. Cloning of genomes of p-lactam-producing streptomycetes into P-lactam-producing hngi or vice versa could yield improved antibiotic fermentation processes or even new antibiotics. Because many genes that affect the growth are located in or near the ribosomal gene cluster in E . coli, isolation and sequencing of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

51

the ribosomal gene cluster of antibiotic-producing organisms may yield information pertinent to the regulation of antibiotic synthesis. Ribosomal genes are present in abundant copies in any organism and provide a vector that can be used to integrate any DNA sequence at many sites on the chromosome of the corresponding microbe. D. RECIPROCAL GENETICSOF SECONDARY METABOLISM

1 . Gene lsolation Methodology is available for the isolation of genes whose level of expression changes in response to the environment. Such genes are involved in secondary metabolism and are heavily transcriptionally regulated. Because these genes are not essential for growth, mutants in them cannot be isolated by conventional genetics. However, these genes can now be isolated and their transcriptional controls dissected. Genes, such as his-3 of yeast, that are constitutively expressed are not the best for isolation by this approach. The highly transcriptionally regulated sequences would be useful for manipulating other sequences to obtain a high level transcription of genes of commercial significance. There may not necessarily be any mutants available in secondary metabolism genes. These genes can be located, however, without tagging by mutation. DNA sequences that have abundant mRNA present in the cell in response to environment are selected. With this in mind, St. John and Davis (1979)looked for genes in Saccharornyces cerevisiae that responded to galactose. They isolated DNA sequences that were complementary to the RNA of galactose-grown S. cerevisiae but had no homology to the RNA of S. cerevisiae that had been grown on another carbon source. Rapid differential screening methods allowed easy isolation of such DNA sequences. It was not necessary to purify mRNA for this purpose, as crude extracts could be used. Total RNA was isolated from yeast grown on lactose, and another preparation of total cellular RNA was obtained from yeast grown on acetate. Both RNA preparations were labeled with 32P.Two replicas of the total genome DNA of many clones of yeast were made. One replica of each clone was hybridized with lactose RNA and the other one with glucose or acetate RNA. The clone that hybridized with lactose RNA and not with glucose RNA was the one of interest and had all the genes responsible for galactose metabolism, which are clustered. There are other genes that are not involved in direct catabolism of galactose but are inducible by galactose (a-glycosylase). These genes have also been isolated by this approach. Also, large sections of DNA belonging to the galactose metabolism region of chromosome have been cloned in a plasmid.

52

VEDPAL SINGH MALIK

2. In Vitro Mutugenesis and Chromosome Manipulation Rare mutational events down to can be screened for by inserting desired DNA fragments into bacteriophage h (Struhl and Davis, 1980; Struhl et al., 1980). The genes can also be mutagenized in vitro before they are put back into the chromosome. A deletion was made in the middle of h i s 3 gene by cutting with restriction enzyme, re-ligating, and then transforming yeast from His+ to His- to inanipulate the chromosome. This has been achieved by inserting the mutant gene in a vector that has a selectable marker, such ura-3. For example, transformants are selected for Ura-3+ and then screened for H i s X . If ura-3 is from a different microbe, the homologous recombination event at the ura-3 locus can be ignored. Most homologous recombinants occur at h i s 3 by forming duplicated unstable structures, giving His-3- and Ura-3+. After 10 generations, 1%of the cells lost the vector by homologous recombination, leaving the altered sequence and removing the wild type and vector. In this way, a genetic system based on homologous recombination and in uitro manipulation of DNA can be built where a genetic system did not exist. All kinds of mutants can be generated without examining the phenotype. Manipulation is termed “reciprocal genetics,” as the phenotype is identified after the genotype has been constructed. DNA of unknown function can be mutagenized and integrated into the chromosome, and the phenotype of the transformant characterized. This allows engineering of mutants without prior knowledge of phenotype and may have direct immediate application for manipulating genes involved in commercially important antibiotics. Three genes are needed for galactose metabolism: transferase, epimerase, and kinase. Accordingly, deletions were made in vitro in these genes, and the constructed genes were ligated to plasmids and put back into yeast as a duplicated structure. These strains were cultured until the vector did not spontaneously excise out at a detectable frequency. A method of scoring the rare excision event utilized a different vector (YRP15), which has tyrosinetRNA suppressor gene as a selectable marker in addition to the ura-3 gene (Struhl et al., 1979a). Only those transformants will grow on media of high osmotic strength that have lost the tyrosine-tRNA suppressor, whereas those containing suppressor tRNA do not grow because of a change in the cell wall (Thomas and James, 1980). This selection against the cells that contain tyrosine-tRNA suppressor allows detection of very rare events. By selecting for Ura-3, one obtains transformants that have received the mutant genes, hut by selecting against suppressor tRNA those transformants are selected where vector and suppressor tRNA have been excised. Such clones can be scored if they have replaced mutant gene for the wild type. This methodology of reciprocal genetics, developed by Davis, can be

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

53

applied to a study of the genetics of secondary metabolism, because secondary metabolism is transcriptionally regulated and responds to the environment.

VI. Control of Secondary Metabolism Microbial productivity is exploited to a maximum in commercial fermentations of many metabolites (Woodruff, 1980). The channeling of metabolic intermediates in the desired direction is helped by the knowledge of the regulatory mechanisms and biosynthetic pathways. As mentioned earlier, antibiotic-producing microorganisms generally produce a variety of biologically active molecules with related chemical structure. The shifting of the component ratio, the repression of the synthesis of undesirable components, or the promotion of the yields of minor components could be enhanced by the knowledge of the regulation of the biosynthetic process. For example, originally cephalosporin C was a minor component of the original Cephalospurium strain of Brotzu. Even though the yield of cephalosporin has been raised to an industrially realizable level with brute force (mutagenesis and medium manipulation), the biochemical basis of superiority of most industrial strains is not known. The knowledge of the regulatory biology responsible for cephalosporin biosynthesis would also be of help in designing rational strain improvement programs. During microbial growth, regulatory controls operate at the level of transcription of DNA into RNA, the translation of RNA into polypeptides, and the activity of enzymes. However, in secondary metabolism and sporogenesis, additional specific control signals are superimposed on those regulating microbial growth and general metabolism. Several such signals could regulate transcription of genes for initiation of secondary metabolism:

1. Like vegetative genes, promoters of the secondary metabolism-specific operons could be available for transcription, but the specific RNA polymerase has to be altered in a manner so that it could transcribe these genes. The preferential inhibition of sporulation and antibiotic synthesis by the polypeptide antibiotic netropsin and by ethidium bromide suggests that the DNA composition of promoters and other controlling elements of genes involved in antibiotic synthesis and sporulation may be different from that for log phase genes (Keilman et al., 1976; Rogolsky and Nakamura, 1974; Sankaran and Pogell, 1975). If the promoters of genes governing secondary metabolism are indeed different from promoters of the genes ordinarily recognized by the vegetative

54

VEDPAL SINGH MALIK

RNA polymerase holoenzyme, the RNA polymerase may be modified to accommodate the promoter recognition specificity of the enzyme that transcribes genes responsible for secondary metabolism. However, if the promoters for secondary metabolism and vegetative genes are identical, then RNA polymerase could be altered to interact with putative regulatory factors that allow RNA polymerase either to bind to the promoters of the genes involved in secondary metabolism or that permit RNA polymerase to read through the blocks presented by negative effectors or special termination sites in genes that govern secondary metabolism. If the vegetative enzyme only abortively initiates RNA synthesis from genes determining enzymes involved in antibiotic synthesis, mechanisms similar to release from termination (Roberts, 1976) or attenuation could affect regulation of secondary metabolism. 2 . Promoters of these genes could be masked by some negative regulatory protein such as a hypothetical repressor. 3. The amount of positive regulatory effectors such as CAMP or ppGpp could be limiting, and in conjunction with a positive regulatory protein, some effectors (e.g., ppGpp) could be used to alter the capacity of these promoters when the repressor has been removed. These factors could be metabolic products that interact with RNA-binding proteins similar to the CAMP-CAPsystem involved in the regulation of the lac operon in E . coli or of glutamine synthetase. 4. Regulation may also occur at the level of translation, involving leaders, attenuators, terminator, and antiterminator sequences in mRNA. 5. Regulatory controls that affect the accumulation of starting precursors for secondary metabolites can indirectly affect initiation of secondary metabolism. The starting precursors have to be available at the time of elimination of the controlling mechanism. 6 . In parallel to synthesis, resistance development to autotoxic metabolites is regulated in the producing organism. 7. Metabolite transport and cellular permeability are also controlled so that high concentrations of secondary metabolite could accumulate outside

the cell.

Practically no experiments have been done that could identify the type of transcriptional and translational apparatus that exists in cells actively engaged in secondary metabolism. Two types of transcriptional apparatus may exist simultaneously. One would transcribe log phase genes and the other would be capable of transcribing genes specific for secondary metabolism and sporulation. To gain an understanding of specific functions required for antibiotic formation, mutants that are resistant to antibiotics with defined site of specificity (streptolydigin, streptovaricins, rifampicin) and that grow normally but are aEected only in formation of secondary metabolism should be selected.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

55

Some mutants may have a genetic lesion at a specific stage in initiation of secondary metabolism. Study of the biochemical basis of such mutations may lead to an understanding of the role of RNA polymerase in secondary metabolite formation.

A. GROWTH-LINKED SUPPRESSION Although biphasic fermentations are common, they are not a sine qua non for secondary metabolism.

VININC(1979)

In 1961, Bu’Lock promoted the theme that many microbes excreted complex organic molecules particularly during stationary phase and not during active growth. In view of the different biosynthetic activity in the successive growth phases, the term “trophophase” has been coined to characterize the period of active cell multiplication and synthesis of primary metabolites. “Idiophase” is correlated with the synthesis of secondary metabolites when active growth is ceased (Bu’Lock, 1961, 1967). This division of growth into trophophase and idiophase brings some order, but it is an oversimplification of the phenomena taking place during the cell’s growth cycle. During the early part of this century, Natural product chemists studied secondary metabolism of fungi. As mentioned earlier, these fungi were usually grown as surface cultures. Stationary cultures develop as surface mycelial mats that cover the entire surface of the medium and are fully exposed to atmospheric oxygen, whereas the lower, partially anaerobic, surface is in direct contact with the growth medium. The kinetics of such growth are different and more complex than the kinetics in shaken cultures. There is often a heterogeneity of metabolic environment in fungal cultures. On the other hand, shake cultures of many streptomycetes and fungi develop as a uniform mass or as pellets of mycelium but are still not completely homogeneous. However, shake cultures were not routinely studied, and the careful experiments that reveal relationships between growth and secondary metabolite formation were not performed. As knowledge of the biochemistry of inicrobiol growth becomes more complete, the data support the view that the relationship between growth and secondary metabolite formation is not quite as simple as originally thought by Bu’Lock (1961) and other naturalproduct chemists. Although the synthesis of some secondary metabolites is an example of the distinct sequence of trophophase and idiophase (Bu’Lock, 1961; Gaworowska et al., 1975), synthesis of many other secondary metabolites does not follow a distinct biphase growth pattern. Most of the chloramphenicol, etamycin, and 6-methylsalicylic acid are produced during the active growth period. Synthesis of rifamycins begins in the early logarithmic

56

VEDPAL SINGH MALIK

growth phasc of Nocardiu mediterranei, and addition of barbital to the culture medium prolongs the logarithmic growth phase and, at the same time, stimulates the synthesis of the antibiotic about twofold (Ruczaj et al., 1972). Synthesis of secondary metabolites also overlaps growth phases in many cultures that are slowly growing in nutritionally poor, chemically defined medium. The induction of upecific enzymes for secondary inetaholism is attributcd to removal of an override mechanism that is operational during the preceding balanced and coordinated growth. The ainidinotransferase in streptomycin fermentation, the acyl transferase and the phenylacetate activating enzyme in penicillin production, arid both enzymes in gramicidin S production are known to be produced in the later part of the growth phase. Phenoxazinone synthetase, involved in actinomycin biosynthesis, is not produced until after growth (Martin and Demain, 1978). Even though the formation of secondary enzymes is generally repressed during the logarithmic growth and is derepressed during suboptimal or stationary growth, and even though their synthesis is controlled by regulatory mechanisms, initiation of secondary metabolism frequently coincides with the exhaustion of some major ingredient of the growth medium and cessation of growth. Penicillin production by Penicillium chrysogenurn 8176 is most pronounced after the exhaustion of both glucose and acetate (Jarvis and Johnson, 1947), and synthesis of anthraquinone pigments of Penicilliuin islariclicuiri begins after the exhaustion of the nitrogen source (Gatenbeck and Sjoland, 1964). Synthesis of ergot alkaloids by Claviceps purpuren and of candicidin by S. griseus begins after all the phosphate has been utilized pining and Taber, 1979; Martin, 1978). A limited supply of oxygen necessary for oxidative metabolism may also induce antibiotic formation (Seddon and Fynn, 1973; Flickinger and Perlman, 1979). Medium that causes slow growth or depletion of an essential nutrient from a rich medium that supports rapid cell proliferation also cause disruption of integrated cellular regulatory mechanisms. This disruption is then followed by relaxation of the growth-associated overriding mechanism that suppresses secondary metabolism. Borrow et al. (1961)have tried to define the factors that initiate secondary metabolism in shake cultures of Gibberella fujikuroi. They have designed media that allow gibberellin production to commence upon exhaustion of any one of several ingredients. They found that gibberellin production occurs during a certain phase of growth of G.fujikuroi, and they defined the following four growth phases (Borrow et n l . , 1964a,b). 1. Balanced phase. This is the period of rapid growth and nutrient uptake that begins at the time of inoculation and ends at the exhaustion of the first

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

57

nutrient. Cell multiplication and most mycelial mass increase occurs until nitrogen, carbon, phosphorus, magnesium, or some other nutrient has been exhausted from the medium. 2. Storage phase. Cell proliferation stops at the beginning of this phase. However, unutilized residual carbon source is converted into mycelial fats and carbohydrates, resulting in increased mycelial dry weight. Other nutrients are consumed and the production of gibberellins begins. 3. Maintenance phase. Constant and maximum mycelial dry weight characterize this phase. Carbon uptake and gibberellin production continue. 4 . Terminal phase. Carbon source has been utilized and mycelium depleted of internal fat and stored carbohydrate. Reserve materials break down, resulting in protein turnover and mycelial lysis. Taber and Vining (1963) have shown that accumulation of ergot alkaloids

by Claviceps purpurea occurs during a prolonged transition phase that is characterized by the accumulation of nitrogenous but not carbyhydrate stor-

age material. This phase falls between the balanced and storage phase of Borrow et al. (1964b), where magnesium and phosphorous were limiting nutrients. Mycelial proliferation continues, but cellular composition changes from that of the balanced growth phase. During growth in some media that do not support alkaloid production, cells pass from balanced into storage phase and store carbohydrates, polyols, and lipids (Spalla et al., 1978; Udvardy, 1980). Vining and Taber (1979) postulated that during logarithmic growth, an unknown growth-linked override mechanism represses the synthesis of the enzymes responsible for the formation of ergot alkaloid by Claviceps purpurea. Ergoline is formed from tryptophan, mevalonic acid, and a methyl group derived from methionine (Maier et al., 1980). Tryptophan plays both a regulatory and a precursor role in this biosynthesis (Otsuka et al., 1980). The former, but not the latter, role can be mimicked by thiotryptophan, which induces dimethylallyltryptophan synthetase, the first pathway-specific enzyme of ergoline biosynthesis. During rapid growth, tryptophan normally accumulates in the mycelium, but the induction of enzymes by accumulated amino acid does not occur until phosphate depletion disrupts growth, thus culminating in ergoline alkaloid production. At this time, some growth-linked override regulatory mechanism ensures that the transcription of genes involved in ergoline biosynthesis is no longer prevented. A high growth rate may alter the amount of some regulatory molecules (e.g., a highly phosphorylated nucleotide) that participates in transcription. Transcription of genes involved in alkaloid biosynthesis may not begin even in the presence of inducing levels of tryptophan, which has probably removed the repressor. It is possible that when exhaustion of nutrients (such as phosphate) reduces the growth rate and adjusts the intracel-

58

VEDPAL SINCH MALIK

lular concentration of a regulatory molecule, the overriding regulatory mechanism, which is growth rate-linked and represses induction of ergoline biosynthesis, is lifted, and genes specific for ergoline synthesis are expressed. An alteration of RNA polymerase may also be one of the pleiotropic controls that govern the expression of genes specific for secondary metabolism. The case of a single secondary metabolite would not be a sound base for attempts at making a model of regulation. Accumulating experience with some of the better-studied systems, such as chloramphenicol, ergot alkaloid, f3-lactarn antibiotics, and actinomycin D biosynthesis, have to be accommodated. There is heterogeneity in these systems. What appears to be the same secondary metabolism of Bu’Lock (1961) may turn out to have different genetic and biochemical bases in different classes of compounds. A thorough examination of the control of many other secondary metabolites may exhibit the same kind of heterogeneity.

B. MULTIVALENTINDUCTION BY PRECURSORS Besides the diversity of their chemical structure and biological activity, secondary metabolites are formed from intermediates or end products of primary metabolic processes. Therefore, their biosynthetic origin could be a basis for examining their regulation. When the grow-linked override mechanism is lifted, the synthetases of secondary metabolism can be produced. However, levels of intracellular substrates for the synthetases determine their activity and rate of secondary metabolite formation. For example, 6-methylsalicylic acid was present in the growing cells of a patulin producer, but 6-methylsalicylic acid was not formed until growth ceased (Bullock et al., 1975). This inactivity of synthetases could be attributable to a limiting supply of substrates and cofactors required for the reaction. A schematic outline of the relationship between primary and secondary metabolism is depicted in Fig. 2. It can be seen that the branch-point intermediates (acetyl CoA, shikimate, malonate, mevalonate, a-aminoadipate) or end products (cysteine, valine, tryptophan, pentose) are usually starting precursors of secondary metabolism (Drew and Demain, 1977). For example, chorismate is a precursor not only of aromatic amino acids and the vitamins but also of chloramphenicol and pyocyanin (Malik, 1979b). Malonyl-coenzyme A is a branch-point intermediate between fatty acids and griseofulvin, tetracycline, patulin, and cycloheximide. Mevalonate is an intermediate that can be channeled either to sterols or to the gibberellins, helvolic acid, fusidic acid, p-carotenes, xanthophylls, terpenes, and ergot alkaloids. a-Aminoadipate is the precursor of lysine, penicillins, and cephalosporins. Acetolactate can also be a precursor of primary and secon-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

Neomycin Gentamycin Kanamycin Tobramycin Ribostamycin Paromomycin Streptomycin Spectinomycin

-t

DNA, RNA

*

Hexose phosphate

Pentose phosphate

-

Tetrose phosphate

Shikimate

Perimycin

Pyocyanin Tetracycline

Serine c -Phophoglycerate

Penicillin

Cephamycin

I

I

rcysteine

Phosphoenolpyruvate

I

I

/

f

Valine c -Pyruvate

I

59

Novobiocin Anthramycin Sibiromycin Lincomycin Rubradirin

4 \f Proprate

Rifamycin Streptovaricin

Psilocybin Lysergic acid Ergoline

Pyrrolnitrin lndolmycin

Cephalosporin

Phenylalanine

1

7

Gliotoxin Maldnate (Malonyl.CoA)

1

Polyketides

'

4

wKetdutarate

1

Showdomycin Oxazinomycin Formycin Pyrazofurin

J

I \

ltaconate

lsoprenoids Ergosterol Cholesterol Gibberellin

Oosperin Orsellinic acid 6-Methylsalicylate Patulin Griseofulvin Aflatoxin Erythromycin Tetracycline Rifamycin Streptovaricin Streptolydigin Cycloheximide

C.c.r,,,rl I.-."._

Carot enoid Trisporic acids

FIG.2. Relationships between primary and secondary metabolism.

dary metabolites. Many secondary metabolites are produced by branched pathways: one branch going to the secondary metabolite, the other branch going to a primary metabolite such as an amino acid. Intermediates of branch pathways could be toxic to the organism if they accumulated in high concentrations intracellularly. Elaborate regulatory mechanisms have evolved that prevent such branch pathways from hnctioning when the end product of the branch has increased up to a certain level. When several metabolites are derived from branches of a common pathway, mutation in one of the branches often leads to the overproduction of the end product of an interconnecting branch. Regulatory mechanisms that control the synthesis of these key branchpoint intermediates are important for increasing the production of secondary metabolites. Common regulatory mechanisms probably control steps early in the branching sequence from primary metabolism. Under growth conditions leading to the accumulation of certain key branch-point intermediates

60

VEDPAL SINGH MALIK

above normal levels (when enzymes of primary metabolism have been completely saturated), the accumulated substrate induces secondary enzyme systems only if the overall override mechanism that is associated with growth has been lifted. Various degrees of nutritional limitation coincide with the cessation of growth and relaxation of the override mechanism. Simultaneously, primary metabolites and intermediates accumulate because many pathways utilizing them slow down and their synthesis is only poorly controlled by either feedback inhibition or repression. In addition, ATP could accumulate because it is not needed for the energy-requiring growth processes. Glucose catabolites, such as acetyl-CoA, accumulate and are not h r t h e r utilized for primary metabolism. ATP inhibits citrate synthase and other enzymes that metabolize the products of glucose metabolism. Under these circumstances, the growth-associated override mechanism is lifted and acetyl-CoA is channeled toward fatty acid or polyketide synthesis by inducing subsidiary pathways. 1. Acetate Polyinalonate Pathway

In 1893, Collie suggested “the manner in which the group -CH2-CW (keten) can be made to yield, by means of simplest reactions, a very large number of interesting compounds; the chief point of interest being that these coinpounds belong to groups largely represented in plants.” Even though Collie’s ideas with respect to the synthesis of terpenes, fatty acids, and aromatic compounds were correct, his work was ignored for more than two decades. In 1919, Raistrick and Clark wrote, “So long ago as 1893 and, more recently, in 1907, Collie, who proposed the term polyketides for the series of compounds containing -CH2COgroups, pointed out the importance for biological chemistry of this class of compounds. These observations do not seem to us to have received from the biochemists the consideration that they deserve.” In 1947, Lipmann discovered coenzyme A, and in 1951, Lynen identified the active form of acetate as acetyl coenzyme A. By this time, availability of radiotracer has helped the biochemists to establish the role of acetate in the biosynthesis of steroids, cholesterol, fatty acids, and other reactions of primary metabolism. Based on the known importance of acetate in the biosynthesis of fats and terpenoids, Birch and Donovan (1953) examined the oxygenation pattern of many natural aromatic products. They found that the position of the oxygen atom indicated that the aromatics were assembled by cyclization of a polyketoinethylene acid, CH,(COCH,),COOH; polyketomethylene acid could be formed by head-to-tail condensation of acetate units. In 1955, Birch et al. provided the first experimental proof of the polyacetate hypothesis. Radioactive 6-methylsalicylic acid was prepared by feeding CH,COOH to cultures of Penicillium griseofulvuni. The labeling

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

61

pattern of isolated 6-methylsalicylic acid as determined by chemical degradation supported the polyacetate hypothesis of polyketide synthesis. Polyketides are all derived from a P-polyketide chain. The polyketide chain consists of acetate or propionate units. It is now known that in fungi the polyacetate pathway is, in fact, an acetate polymalonate pathway. AcetylCoA in the starter position is condensed with malonyl-CoA, accompanied by loss of CO,. Malonate is derived by carboxylation of acetate. Polyketides in which methylmalonyl (propionate) units are the starter units and are extensively reduced [as in macrolides (erythromycin)] are characteristic of Streptumyces. In higher plants, aromatic amino acids serve as starter units of flavanoids and other polyketides. Flavanoids are synthesized in plants by condensation of one acetate-malonate-derived monobenzenoid nucleus with one shikimic acid-derived monobenzenoid nucleus. Polyketide antibiotics can be divided into two groups: (1) Antibiotics in which the carbon skeleton is derived from acetate, propionate, and butyrate (e.g., nanaomycins); (2) Antibiotics in which sugars and amino acids contribute to the carbon skeleton (e.g., spiramycin, tylosin). Polyketides and the common fatty acids have a common biogenic constitution because both are chains of acetate-derived subunits. Because of the participation of special carrier proteins, intermediates and end products are not in equilibrium with other cell components. For instance, acetoacetylCoA that is involved in fatty acid synthesis does not equilibrate with acetoacetyl-CoA that is intermediate in orsellinic acid synthesis because both are bound to their respective synthetases and are not free in cytoplasm. However, in streptomycetes antibiotics, the primer propionyl-CoA condenses with methylmalonyl-CoA. As a result, 3-carbon units are added, resulting in methyl groups attached to the growing polyketide chain. Aglycone (erythronolide) of erythromycin is synthesized by this route, which involves condensation of propionate and methylmalonic acids. Malonyl-CoA serves as a primer for tetracycline biogenesis and supplies the C-2 carboxyl group. The remaining carbon atoms of the ring may also come from either the polyketide route or chorismate. The C-6 methyl group is introduced prior to ring closure and is donated by methionine. The nitrogen atom at C-4 arises via transamination. The C-6 hydroxyl and C-5 hydroxyl are derived from molecular oxygen. Malonyl-CoA for the biogenesis of chlortetracycline in S. aureofaciens may not be derived from acetate but from oxaloacetate (Hostalek, 1978). Acetyl-CoA carboxylase is not present in high-chlortetracycline-producing strains during antibiotic production. The low activity of acetyl-CoA carboxylase in S. aureujkciens during chlortetracycline production is correlated with low activity of pyruvate kinase and pyruvate dehydrogenase complex. This indicates that the conversion of phosphoenolpyruvate to acetyl-CoA is

62

VEDPAL SINGH MALIK

suppressed when chlortetracycline is being produced. Phosphoenolpyruvate carboxylase is very active, yielding oxaloacetate, which is further channeled toward malonyl-CoA synthesis, a precursor required for chlortetracycline biogenesis. A wide variety of both aromatic and nonaromatic molecules are produced by various microbes via acetate-malonate condensation. Different enzyme complexes exist to form different oligoketide-derived products with different affinities for the acetyl and malonyl moieties. Relative lack of NADPH during certain phases of growth might favor the formation of oligoketides (orsellinic acid, 6-methylsalicylic acid, alterionol) at the expense of fatty acids, which require substantial reducing power in the form of NADPH for their synthesis. In the polyketide route, the keto groups generated during condensation of malonate and starter molecule are not reduced as in fatty acid biosynthesis. Thus, the concentration of reducing cofactor (NADPH) could be regulating diversion of acetate to fatty acid or polyketide. Gatenbeck and Hermodsson (1965)examined the possibility of NADPH as a regulatory factor involved in determining the fate of acetyl and malonyl donors in cell-free extracts of Alternuria tenuis. In the present of NADPH, utilization of acetyl-CoA was large for lipid synthesis relative to alternariol formation. Enzymatic reactions involved in the genesis of secondary metabolites compete for the same coenzymes and ions that catalyze basal metabolic reactions. Subtraction of these compounds from the common pool d e c t s other processes in which these coenzymes and ions take part. For instance, synthesis of rifamycins is accompanied by a distinct shift in the oxidizedl reduced nicotinamide adenine dinucleotide (NADINADH) and pyruvate/ lactate ratios toward the oxidized forms. This suggests a requirement for hydrogen equivalents in rifamycin synthesis and indicates an effect on the oxidation processes in N . meditmunei (Ruczaj et al., 1972).The occurrence of rifamycins in forms showing different degrees of oxidation of the molecule suggests that rifamycin may play the role of an oxidoreduction agent in the metabolism of the microorganism that produces this antibiotic. Hostakek et al. (1969) have called attention to the deficiency of acetate units in the synthesis of oligoketide antibiotics. These investigators have shown that intense energy production is unfavorable for the synthesis of tetracyclines by S. aureofuciens. High activity of the enzymes of the citric acid cycle oxidize acetate and diminish the pool of acetate, a building block of oligoketide antibiotic tetracycline. Roszkowski (1972) correlated carboxylation of acetate and propionate with polyene biosynthesis. In a high-producing strain of Streptomyces noursei var. polijungini, the total pool of acyl-CoA was fivefold greater than in a

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

63

low producer, when cells were grown on carbohydrates or lipids. Mutants that produce increased amounts of polifungin have twice the activities of acetyl-CoA and propionyl-Cod carboxylases as compared to the parent. Carboxylase activity toward both acyl-CoA derivatives and polyene production also correlates in low-producing strains. Rafalski and Raczynska (1972, 1973) also observed that synthesis of the nystatin-type antifungal antibiotic polifungin and of lipids in S. noursei var. polijungini is correlated with increased activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase. These enzymes produce malonate and methylmalonate, which are units for elongation of oligoketide. Mutants blocked in the fatty acid synthesis pathway leading from malonyl-CoA should further increase the flow of malonyl-CoA to oligoketides, increasing their yields and altering cell membrane permeability properties simultaneously (Malik, 1979a). Catabolism of exogenous fatty acids via P-oxidation increases the cellular pool of acetyl-CoA. Fatty acid esters also reduce further oxidation of acetylCoA by inhibiting citrate synthetase. A high supply of carbohydrates also leads to elevated levels of acetyl-CoA, which is channeled toward formation of polyketides. Glucose inhibits fatty acid catabolism by catabolite repression. Both tylosin synthesis and oleic acid oxidation were inhibited simultaneously by glucose, but enzymes involved in the synthesis of mycarose, the sugar moiety of tylosin, were not affected. Cerulenin is an inhibitor of the condensing enzyme that condenses acetyl-ACP and malonyl-ACP to forin P-ketoacyl-ACP, a precursor of fatty acids. The initial enzyme involved in the biosynthesis of polyketides is similar to the condensing enzyme of fatty acid biosynthesis as biosynthesis of polyketides is also inhibited by cerulenin. Mutant strains that are resistant to cerulenin may overproduce polyketides. As a matter of fact, daunorubicin, an anthracycline antitumor antibiotic, is overproduced by a ceruleninresistant streptomycete mutant (McGuire et al., 1980).

2 . Shikimate and Chorismate Although the acetate polymalonate pathway is prominent in eukaryotes, the shikimic acid pathway is generally used for synthesis of secondary metabolites in prokaryotes. Pyocyanin, candicidin, corynecins, chloramphenicol, and aromatic amino acids are synthesized by the shikimic acid pathway. In 1954, Tatum and Gross noted that a mutant of Neurospora that was blocked in the conversion of deh ydroshikimate to shikimate accumulated protocatechuate. Accumulation of dehydroshikimate induced a secondary pathway channeling into the formation of protocatechuate. Another example of a fungal secondary metabolite derived from shikimate is the

64

VEDPAL SINGH MALIK

terphenylyuinone volucrisporin. Chandra et al. (1966) showed that ni-hyclroxyphenylpyruvateis the intermediate between shikimate and volucrisporin. Ansamycins are characterized by an aliphatic, oligoketide-type bridge, which connects two different positions to an aromatic moiety (e.g., streptovaricins, rihamycins). The 7-carbon amino unit of the naphthoquinone part of rifamycins is derived from a shikimic acid-like precursor. Mutants of AT. mediterruiiei, one blocked in the transketolase and another in shikimate kinase, accumulate o-ribulose and shikimate, respectively, in amounts equivalent to the rifamycin B produced by the parent strain. This suggests that primary metabolites (like ribulose) are channeled in equimolar amounts to synthesis of secondary metabolites. By manipulating regulation of primary pathways, one could, therefore, increase yields of antibiotic and other fermentation products. Synthesis of chloramphenicol by S . venezuelue is subject to control by integrative regulatory mechanisms at several levels. End-product inhibition modulates the amount of chorismic acid entering into the chloramphenicol branch of the aromatic pathway. The first enzyme (3-deoxy-Darabinoheptulosonate phosphate synthetase) is neither repressed nor feedback-inhibited by the intermediates and end products of the pathway. Synthesis of none of the other enzymes involved in aromatic amino acid synthesis is repressed by these amino acids or the intermediates of the pathway. L-Phenylalanine and L-tryptophan inhibit prephenate dehydratase and anthranilate synthase activities, respectively. Thus, induction of chloramphenicol synthesis could occur when the pool of chorismate at the branch point is diverted from the multiple branch pathways leading to the various aromatic metabolites. This type of regulatory pattern is called “multivalent induction” because the end products of multiple branch pathways control the level of chorismate, the intermediate at the inducing branch point (Malik, 1980a). Numerous chloramphenicol analogs that could provide tools for genetic and biochemical analysis of the system have been synthesized. The mechanism of chromosome transfer in S . venezuelae has been revealed, and circular genetic maps have been constructed (Francis et a l . , 1975). This organism also harbors a plasmid that can be used as a vector for transporting recombinant DNA molecules (Malik and Reusser, 1979). Transducing phages for this streptomycete have also been reported. Chloramphenicol is produced in well-defined medium, and the total RNA isolated from cells that are engaged in chloramphenicol synthesis can be used as probe to select genes that are expressed during chloramphenicol production. Addition of aromatic amino acids stimulates chloramphenicol synthesis, but the amount of chloramphenicol produced is also limited by the sensitiv-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

65

ity of the producing organism to the produced antibiotic. Therefore, resistance of the organism to the antibiotic should first be increased before other approaches to yield improvement are initiated. Chloramphenicol is commercially produced by chemical synthesis. The cost of chloramphenicol may be decreased if a successful fermentation for its production could be developed. The fact that production of chloramphenicol parallels growth of S . venezuelae may be exploited to develop a continuous culture commercial fermentation for chloramphenicol. Tryptophan is an essential amino acid with a 50-million-dollar market. Yield of tryptophan by microbial fermentation has been poor. However, many microbes that produce chorismate-derived metabolites may be manipulated to excrete tryptophan. Such microbes have evolved with mechanisms to accumulate intracellular pools of chorismate, which could be diverted toward the synthesis of tryptophan, eliminating secondary metabolite production. Several chorismate-derived metabolites (e.g., candicidin, rifamycin, and ergot alkaloids) are being produced in quantities of up to 10 gmfliter in commercial fermentations. Organisms that excrete ergot alkaloids could be manipulated to develop a commercial tryptophan fermentation.

3. Mevalonate and Isoprene Unit The number of secondary metabolites derived from mevalonate is rapidly increasing. In addition to its role as a precursor of various secondary metabolites, mevalonate is often a precursor of side chains of one or more isoprene residues. Two commercially important mevalonate-derived fungal products are the gibberellins and sterols (Rose, 1980).

4 . Amino Acids as Precursors Amino acids that are derived from the tricarboxylic acid cycle intermediates form the starting points for a number of alkaloids, peptides, and p-lactam antibiotics.

a. Peptides. Peptide antibiotics (gramicidin, tyrocidine, valinomycin, bacitracin) are synthesized by multifunctional enzymes utilizing the protein thiotemplate mechanism. Such multienzyme complexes have high reaction efficiencies as a result of cellular compartmentation of intermediates. A growing peptide chain is. synthesized by a phosphopantotheine carrier in a series of transpeptidation and transthiolation steps (H. Kleinkauf, Technische Universitat, Berlin). Peptide bond formation occurs without tRNA, mRNA, and ribosomes. The amino acid sequence in the peptide antibiotic is dictated by the specificity inherent in multienzyme complexes. The longest peptide synthesized by such a mechanism in vitro is alamethicin, which consists of 20 amino acid residues. However, the biosynthesis of

66

VEDPAL SINGH MALIK

cyclic decapeptide antibiotics, gramicidin S and the tyrocidines, has been well worked on. Two multifunctional polypeptide chains known as the heavy and the light enzyme are involved in the biosynthesis of gramicidin S: cyclo(D-Phe-Pro-Val-Orn-Leu).Neither of these two gramicidin S synthetases can be dissociated into subunits by protein-denaturing agents, which suggests that several functional domains on a polypeptide chain are covalently bound. Gramicidin S synthetase probably carries 24 different catalytic functions. The activation center for proline and the transfer region for phenylalanine may have regulatory functions affecting the biosynthesis of gramicidin S. Binding of proline stimulates the other reaction centers of the multienzyme, thus regulating the efficiency of the biosynthesis. This effect is strongly increased if phenylalanine is transferred to the initiation site of the synthetase by the light enzyme. Studies on the activation of amino acids show that binding of substrates is random in formation of the aminoacyl adenylates, except in the case of ornithine, where evidence for an ordered mechanism has been obtained. Rate of formation and stability of enzyme-bound adenylates is lower than in corresponding aminoacyl-tRNA ligases.

b. P-Lactarn Antibiotics. All P-lactams have common amino acid precursors (Fig. 3). Both penicillin and cephalosporin ring systems are synthesized from L-cysteine and L-valine. Penicillin N is produced in all cephalosporin C fermentations. However, the reverse is not true. Relative yields of penicillin N and cephalosporin C are changed inversely by varying aeration of C. acremonium or by addition of methionine or carboxymethyl-L-cysteine to washed mycelial suspensions. Hydrophobic benzylpenicillin needs La-aminoadipic acid (L-MA) as an obligatory “precursor,” which is finally exchanged with the coenzyme A ester of the side chain acid. 6-Aminopenicillanic acid (6-APA), the penicillin nucleus, is not an intermediate but is formed as a shunt product when no side-chain precursor is present. Isopenicillin N is an intracellular hydrophilic intermediate. The pathway is as follows: L-AAA

+ L-CYS+ L.-AAA-L-CYS

LLD-trlpCptidC’

L-Val

isopiwicillin N

ptwicillin G

+

L-AAA

i 1 1

ph2nylact:tyl COA

In Cephalospurium, the cyclization of LLD-tripeptide to isopenicillin N is stimulated by Fez+.Isopenicillin N is converted to penicillin N by inversion of its L-AAA side chain to D-AAA. Penicillin N is then subjected to ring

1

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM 0-KETOGLUTARATE

EXTRACELLULAR METHIONINE

I

HOMOCITRATE SY NTHETASE

f

HOMOCITRATE

1

cis-HOMOACONITATE

1 1

HOMOISOCITRATE

U-KETOADIPATE

aF d

72 I-

%

PYRUVATE

I

METHIONINE

1

ACID SYNTHETASE

U-~-OIHYOROXYISOVALERATE

1

S-ADENOSYL METHIONINE

1

-1

S-ADENOSYL HOMOCYSTEINE

1

67

KETOISOVALERATE

7

HOMOCYSTEINE

W 0

L.o. AMINOADIPATE

6 ADENYL-0-AMINO AOIPATE

J

\ I

~-IL-u-AMINOADIPYL)-CYSTEINE

n-AMINO-8-ADIPY L-SEMIALOEHYOE

t

l-

[L-VPILII~T I

AMINOADIPY LJ-L-~YSTEINYL-&VA~INE

s-~L-

SACCHAROPINE ISOPENlClLLlN N

PENICILLIN G

DEACETOXYCEPHALOSPORIN C

DEACETYLCEPHALOSPORIN C

1 i

CEPHALOSPORIN C

7-METHOXYCEPHALOSPORI"

FIG. 3. The biosynthetic pathway to p-Iactam antibiotics.

expansion to deacetoxycephalosporin C . Early-blocked Cephalospwium mutants, which do not produce penicillin N or cephalosporin C in fermentation, catalyze cell-free ring expansion. Late-blocked mutants produce only penicillin N and do not catalyze cell-free ring expansion. The ring expansion reaction is stimulated by Fez+,ascorbate, and ATP. The terminal steps of the biosynthetic pathway include the oxidation of deacetoxycephalosporinC by a dioxygenase to deacetylcephalosporin C. The dioxygenase is stimulated by Fez+,ascorbate, and a-ketoglutarate. Deacetylcephalosporin C is acetylated to cephalosporin C (Baldwin et al., 1980; Sawada et al., 1980a,b,c). Recently prokaryotic streptomycetes have been shown to produce not only

68

VEDPAL SINGH MALIK

hydrophilic penicillin N and 7-methoxycephalosporins but several new p-lactams. Total synthesis of 1-oxacephalothin and 1-carbacephalothin establish that sulfur can be replaced without loss of biological activity by oxygen and -CH2-, respectively. Naturally occurring compounds of this type with no sulfur-containing, hsed-ring system may well be produced by streptomycetes. Pathways for the biosynthesis of a-aminoadipic acid differ in fungi and streptomycetes. In streptomycetes, degradation of lysine provides a-aminoadipic acid (Stirling and Elson, 1979) whereas in fungi, a-aminoadipic acid is the precursor of lysine. Two other compounds with the penicillin ring system (8aminopenicillanic acid and isopenicillin N) are produced by P . chrysogenum. However, in addition to producing penicillin N, Cephalospurium species produce cephalosporin C. Cephalospurium species have never been found to produce either the nucleus of cephalosporin C, 7-aminocephalosporanic acid, or any other p-lactam antibiotics that have substituded D-a-aminoadipic acid. Stages in the biosynthesis of clavulanic acid by Streptomyces clavuligerus (Elson and Oliver, 1978) do not fit in the scheme proposed for the biosynthesis of p-lactam antibiotics in eukaryotic fungi (see Fig. 2). The penicillin and cephalosporin precursor, &(a-aminoadipy1)cysteine-valine is not involved in the biosynthesis of clavulanic acid, which lacks a 6-amino group and aminoadipyl side chain and possesses an oxazolidine rather than a thiazolidine or dihydrothiazine ring. Glycerol is incorporated intact into the three p-lactam carbons of clavulanic acid; the remaining five carbons being derived from a-ketoglutarate. These studies with the biosynthesis of clavulanic acid in S. clavuligerus assert that different pathways for biosynthesis of p-lactam ring do exist in procaryotic streptomycetes and eukaryotic hngi. c. Lysine Effect. Regulatory processes that control the supply of basic building blocks exert a direct influence on antibiotic formation. a-Aminoadipic acid is the branch point for lysine and benzylpenicillin biosynthesis. Lysine could inhibit its own synthesis from a-aminoadipic acid, causing a rise in the level of the latter in the cell and diverting it toward p-lactam synthesis. However, a different situation exists in penicillin-producing fungi. When lysine is added to the fermentation medium, homocitrate synthetase is inhibited and repressed. This depletes the intracellular level of a-aminoadipic acid and thereby decreased production of penicillin (Luengo et al., 1980). Because of impermeability of prototrophic Cephalosporium, antibiotic synthesis is neither inhibited by lysine nor promoted by a-aminoadipic acid. However, a lysine auxotroph blocked after a-aminoadipic acid grows well, but because of feedback inhibition of homocitrate synthase, forms no cephalosporin

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

69

(Lemke and Nash, 1972). Addition of lysine to streptomycetes fermentations provides a-aminoadipate and thereby stimulates production of 7-methoxycephalosporins. In a cephamycin producer, S. clauuligerus, the activity of the aspartokinase is subjected to concerted feedback inhibition by lysine and threonine. Mutants resistant to lysine analog S -(2-aminoethyl)-~-cysteineproduced from 1.5 to 4 times as much antibiotic as the analog-sensitive parent. The aspartokinase activity of the enzyme obtained from analog-resistant mutants was not inhibited by the concerted effect of lysine and threonine.

d. Valine. The configuration of the valine moiety in p-lactams is D; and valine provides the carbon skeleton of the penicillamine residue of the p-lactam antibiotics. Addition of valine to complex fermentation media does not affect penicillin yield. However, ~-valineincreases the rate of penicillin formation by washed mycelium. The conversion from pyruvate to acetolactate is the initial step in valine formation and is catalyzed by acetohydroxy acid synthetase. This enzyme is subject to feedback inhibition by ~ - v a l i n ein wild-type strains. Acetohydroxy acid synthetase from a high-penicillin-yielding mutant was much less sensitive to feedback inhibition by L-valine. Furthermore, the mutant enzyme had only one binding site for valine, compared to the two binding sites for the ancestral enzyme. Enzyme content in the superior strain was also twice that in the parent (Goulden and Chattaway, 1968, 1969). The specific activity of glutamate dehydrogenase was derepressed in a high-yielding mutant, whereas low-yielding mutants were repressed for the synthesis of this enzyme (Queener et al., 1978). The altered regulation pattern for glutamate dehydrogenase may enhance nitrogen assimilation for cephalosporin C synthesis. An inverse relationship between vegetative mass and cephalosporin C was observed by Queener et al., suggesting that conditions that are best for vegetative development are usually worst for antibiotic production. Superior antibiotic producers of Streptomyces lipmunii lack control in the Ile-Leu region, suggesting that valine synthesis can be a rate-limiting step in antibiotic production (Godfrey, 1973). e. Methionine and Sulfur Metabolism. The effect of the metabolism of sulfur-containing compounds on p-lactam antibiotics has been extensively investigated (Drew and Demain, 1977). Methionine can be used as the sole source of nitrogen or sulfur by P-lactam-producing organisms. Furthermore, methionine exerts a stimulatory effect on the yield of cephalosporin if added during vegetative growth of C. acremonium. Because other sulfurcontaining compounds (such as cysteine), which are efkient precursors of

70

VEDPAL SINGH MALIK

the sulfur atom of cephalosporin, are without stimulatory effect on the antibiotic yield, methionine must also have a regulatory role. Several groups (Drew and Demain, 1977) have used mutants blocked in sulfur metabolism to elucidate the role of methionine in cephalosporin C formation. In an early-blocked mutant that grows on cysteine, methionine, cystathionine, and homocysteine, but not on sulfate, the magnitude of cephalosporin production stimulation was methionine > cystathionine > cysteine. If methionine was stimulating cephalosporin production only as a result of being the precursor of sulfur for the antibiotic, the sulfur amino acids should stimulate cephalosporin C production in the reverse order of that observed by Drew and Deinain (1975c, 1977). This opposite order of stimulation experimentally obtained suggests that cysteine and cystathionine are converted via transsulfuration to methionine, which then exerts the regulatory effect and stimulates cephalosporin C synthesis. To support this point, Drew and Demain (1975~) blocked transsulfuration from cysteine to methionine by mutating an early-blocked mutant in sulfur utilization. This double mutant did grow on methionine but not on cysteine or sulfate and produced little antibiotic in the presence of excess cysteine and low enough levels of methionine just to allow normal growth. This double mutant produced cephalosporin in the presence of excess methionine. Furthermore, a nonsulfur-containing analog, norleucine, could replace excess methionine, supporting the regulatory role of methionine. Methionine may also be the inducer of cephalosporin formation in C. acremonium, but the exact nature of the molecular mechanism involved here is hard to visualize. A methionine auxotroph of C. acreinonium produced cephalosporin yields greater than its parent when supplemented with methionine. A CIBA mutant blocked in the sulfate reduction pathway prior to sulfide formation assimilated more exogenous methionine and synthesized four times more cephalosporin than its parent. High levels of cephalosporin C production with a non-sulfate-utilizing mutant could be a result of its inability to synthesize cysteine, the repressor of methionine permease. Mutants of C. acreinonium that efficiently synthesize cephalosporin C from sulfate have been isolated. One mutant is similar to the cys-3-mutant of Neuraspura crussa in which synthesis of sulfate permease as well as aryl sulfatase is coordinately controlled. This mutant utilized sulfate as effectively as methionine for cephalosporin C synthesis. A natural isolate of C. acremoniuin was derepressed for aryl sulfatase and synthesized cephalosporin preferentially from methionine, deriving sulfur via transsulfuration. Aryl sulfatase repression in the mutant may be attributable to accumulation of sulfide, a corepressor of sulfatase in fungi. Another mutant efficiently utilized sulfate and produced double the amount of cephalosporin produced

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

71

by its parent. Cephalosporin production by this mutant was sensitive to methionine. A methionine synthase is only operative in sulfate-mediated cystathionine formation that is repressed by high concentrations of exogeneous methionine but stimulated by an excess of sulfate. This is why mutants with an operative alternative route produce increasing cephalosporin C from sulfate and have limited tolerance for methionine. The increase in cephalosporin C production from sulfate and methionine sensitivity may both be the regulatory consequences of a single mutational event. A mutant lacking cystathionine y-lyase did not grow on methionine, homocysteine, and cystathionine, but did grow well on cysteine and inorganic sulfur. Methionine did not stimulate cephalosporin production in synthetic medium but decreased the yield to about 20%ofthe antibiotic production of the parent. Furthermore, in the sulfate-supplemented, methionine-free medium, the antibiotic potency of the mutant was lower than the parent. These results demonstrate the importance of cystathionine y-lyase for methionine-stimulated antibiotic synthesis and indicate that cystathionine may be a prerequisite for antibiotic synthesis. The cleavage of cystathionine may induce the transfer of a cysteine moiety into a peptide intermediate involved in p-lactam synthesis (Treichler, 1979). Cystathionine synthesis from inorganic sulfide occurs by two alternative routes. Both of these routes were blocked in a mutant defective in hoinoserine 0-acetyltransferase. This 0-acetylhomoserine auxotroph was resistant to methane selenol. Because of block in the synthesis of 0acetyl-L-homoserine (the common acceptor substrate for both metabolic routes leading to cystathionine), this mutant was unable to synthesize cystathionine from cysteine or inorganic sulfur. However, the mutant grew on methionine, homocysteine, and cystathionine, but not on cysteine or inorganic sulfur. In synthetic medium supplemented with low levels of rnethionine and excess cysteine or sulfate, this mutant did not produce increased cephalosporin. The parent or revertants of the mutant produced cephalosporin C in good yields on addition of excess cysteine or sulfate. This mutant required high levels of methionine to produce cephalosporin C. The parent, but not the mutant, produced considerable amounts of cephalosporin C in media supplemented with cysteine or sulfate. Blocking both routes to cystathionine virtually eliminated cephalosporin C production from cysteine or inorganic sulfur. This decreased antibiotic productivity suggests that cephalosporin is derived through cystathionine by means of sulfide fixation utilizing 0-acetylhomoserine sulfhydrylase (methionine synthase) and cystathionine p-synthase (Treichler, 1979). Simultaneous operation of both routes to cystathionine biosynthesis may have an additive effect on cephalosporin production. To understand this, the other route of anabolic cysteine synthesis was also blocked by mutagenesis of

72

VEDPAL S I N G H MALIK

a strain with impaired cystathionine y-lyase; the parental marker was removed by reversion. These double mutants grew on cysteine but not on inorganic sulfur, methionine, homocysteine, or cystathionine. All these mutants were methionine- and homocysteine-sensitive. This inhibition was only reversed by cysteine. In synthetic media, no difference in productivity was found between mutant and parent when methionine was used as the sulfiir source. The impairment of anabolic enzyme O-acetylserine sulfhydrylase had a positive effect on cephalosporin C production with excess sulfate in the medium. A block in the anabolic synthesis of cysteine must encourage conversion of sulfide to cystathionine by the alternate route. Addition of methionine to the fermentation medium has no effect on penicillin yield in P . chrysogenurn because in penicillin sulfate is reduced into cysteine via cysteine synthetase. However, in cephalosporin, cysteine is derived exclusively from methionine via reverse transsulfuration. Both O-acetylhomoserine sulfhydrylase and methionine synthase, but no O-acetylserine sulfnydrylase (cysteine synthase), were present in cell-free extracts of P . chrysogenurn during penicillin production in sulfate-containing synthetic and complex medium. This suggests that sulfide to cystathionine via hoinocysteine is the main route for optimal p-lactam synthesis in P . chrysogenum. Wild-type Penicillium strains use sulfate for antibiotic synthesis because they utilize this alternative route. Cephulosporiuni acremoniuin possesses potent O-acetylserine sulfhydrylase, the activity of which exerts an inhibitory effect on the operation of the alternate route. Therefore, mutants in this enzyme will utilize an inorganic sulfur source by an alternate pathway. The stimulation or inhibition of the alternate route could be attributable to many factors. A C. acrenzonium mutant with an enhanced potential to utilize sulfate for cephalosporin C production (Komatsu and Kodaira, 1977) exhibited elevated cystathionine P-synthase activity. O-acetylserine sulfhydrylase activities in the parent and the mutant were similar. Mutations enhancing levels of enzymes involved in the alternate route of cystathionine biosynthesis can increase p-lactam yield. The common nonprotein amino acids, such as sarcosine and ornithine, are frequent components of antibiotics. Synthesis of these amino acids follows known pathways. For instance, ornithine, a constituent of bacitracin, is formed during antibiotic production both from glutamate, as intermediate in the synthesis of arginine, and as the degradation product of arginine. Enzymes catalyzing anabolic as well as catabolic production of ornithine from arginine are induced during sporogenesis and synthesis of bacitracin in B. subtilis (Pass et a l . , 1974). The nonprotein amino acid constituents are also limiting in the synthesis of peptide antibiotics, a , y-Diaminobutyric acid and ornithine affect synthesis of colistin and bacitracin, respectively (It0 et a l . , 1970; Pass et al., 1974).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

73

Mutation that prevents ornithine degradation by ornithine 6-transaminase enhances antibiotic production and simultaneously decreases the glutamate and proline pools. Both of these amino acids are formed from ornithine (Pass et al., 1974).

5 . Sugar Derivatives These compounds originate from pentoses and hexoses, which during growth are incorporated into polysaccharides and nucleic acids. During trophophase of P. clirysogenum, glucose is metabolized primarily by the hexose monophosphate shunt, yielding large amounts of NADPH. However, in the idiophase, glycolysis is predominant. The carbon skeleton of glucose is incorporated either intact into aminoglycosides (e.g., streptomycin) (Kirby, 1980)or partially as a glycoside moiety attached to a carbon skeleton derived by another pathway (as in macrolides, e.g., erythromycin). Deregulation of the hexose monophosphate shunt generates from glucose excess intracellular ribose, which is the precursor for nucleosides and aminoglycoside antibiotics. Glucose provides acetate, propionate, and NADPH for the successive reduction steps of the highly oxidized polyketide chain. Glucose is also involved through the pentose phosphate cycle in the biosynthesis of the p-aminoacetophenone moiety of candicidin. In the chlortetracycline-producing strains ATP glucokinase attained peak activity after about 12 hr of incubation and then declined in parallel to the decrease in cellular ATP level. The activity of this enzyme was the lowest during the stationary phase of growth. Glucose-6-phosphate formation showed the presence of an alternate mechanisms of phosphorylation of hexoses that supply building blocks for antibiotic synthesis, i. e., polyphosphate glucohnase, which was present in the culture only during antibiotic production and after ATP glucokinase activity had diminished. Cellular polyphosphate was maximum after the drop in ATP level. This is connected with the shift from the adenylate phosphorylation mechanism to the polyphosphate system. Production culture possessed 10% adenylates of the polyphosphates. In S. coelicolm, uptake and metabolism of arabinose, glycerol, fructose, and galactose were repressed by glucose, cellobiose, mannose, and nonmetabolized 2-deoxyglucose (Hodgson, 1980). Mutants that can grow on glycerol, arabinose, etc., in the presence of 2-deoxyglucose have lost repression by glucose of many sugar-metabolizing pathways. Most mutants that are resistant to 2-deoxyglucose probably are missing glucose kinase, and the corresponding mutations map at two chromosomal locations. A small number of 2-deoxyglucose-resistantmutants utilize glucose and may be altered in a protein involved in carbon catabolite repression. None of the 2-deoxyglucose-resistant mutants overproduced actinorhodin or methyl-

74

VEDPAL SINGH MALIK

enomycin. However, they sporulate potently, suggesting that glucose represses sporulation. Romano and Margiotta (unpublished results) have used the nonmetabolizable analogs 2-deoxy-D-glucose (2-DOG) and 6-deoxy-~-glucose(6-DOG) to study the properties of the D-glucose transport system of Streptomyces griseus MA45. Glucose-grown vegetative cells accumulate both these analogs against a concentration gradient. The transport system has an apparent K , of 24 p M for 2-DOG and 27 p M for 6-DOG and is competitively inhibited by D-glucose with a K , of 6 p M . 6-DOG is accumulated exclusively as the free sugar. 2-DOG is transported as the free sugar but is accumulated intracellularly both as freesugar and as sugar phosphate, the latter as a result of hexokinase activity. Toluenized cells showed ATP-dependent phosphorylation of glucose and 2-DOG, but no phosphoenolpyruvate(PEP)-dependent phosphorylation of these sugars. Thus, these sugars are accumulated by an active transport system and not by a PEP:hexose phosphotransferase system. The transport system is inhibited by respiratory inhibitors such as NaCN and by protonconducting ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone, which prevent both the establishment of a proton motive force and the synthesis of ATP. It is not inhibited by reagents that prevent ATP synthesis only, such as arsenate of N,N’-dicyclohexylcarbodiimide.These data indicate that the glucose transport system is energized by a proton motive force and not by ATP directly. The system is specifically inducible by glucose and shows high specificity in its activity. Thus, fructose- or galactosegrown cells do not transport 2-DOG or 6-DOG. Fructose or galactose do not show activity for the glucose transport system in glucose-grown cells, either as inhibitors of 2-DOG uptake, or as uptake substrates.

c. FEEDBACK INHIBITION AND ENDPRODUCT REPRESSION

Secondary metabolites are often excreted into the environment and rarely accumulate intracellularly. In such situations, excessive extracellular product may not act directly by interacting with the allosteric branching enzymes or regulatory proteins (Vining, 1979). The biosynthetic intermediates or degradation products of the excreted metabolite could be the real regulators of these secondary pathways. Several secondary metabolites are reported to exert controlling effects on their own synthesis. 6-Methylsalicylic acid synthetase, a multienzyme complex, is responsible for the synthesis of 6-methylsalicylic acid (6-MSA) by Penicillium uriticae from acetyl-CoA, malonyl-CoA, and NADPH. Like yeast fatty acid synthetase, it is composed of four identical multifunctional

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

75

polypeptides. Each subunit has a molecular weight of 790,000. Two types of thiol groups (cysteine and cysteamine) and two partial activities (palmityl transferase and dehydratase) are present. The immunological cross-reaction between both 6-MSA synthetase and fatty acid synthetase suggests functional similarity between these two enzymes. Despite the resemblances, they are different proteins coded b y separate genes. Patulin inhibits the total activity and condensation step catalyzed by 6-MSA synthetase. Because polyketide synthesis depends on the formation of malonyl-CoA from acetyl-CoA, it will be affected by the same regulations of the carboxylase reaction that control fatty acid synthesis, including allosteric activation by citrate and feedback control by long-chain fatty acids. Mechanisms of this kind may explain the adverse effects of lipid-antifoams on the yield of griseofulvin and other polyketides. In the chloramphenicol-producing cultures, arylamine synthetase, the branch-point enzyme, is not feedback inhibited but is repressed by increasing concentrations of chloramphenicol (Jones and Westlake, 1974; Malik, 1979b). Because chloramphenicol is excreted into the medium and never accumulates intracellularly, some intermediates of the biosynthetic pathway probably repress transcription of the arylamine synthetase gene. Nakano and co-workers (1974) have shown that synthesis of corynecins, which are structurally closely related to chloramphenicol, was repressed by the p-aminophenylpropanoid intermediate. Addition of tryptophan to the candicidin-producing cultures of S. griseus represses and feedback-inhibits p-aminobenzoic acid synthetase. This curtails the amount of cellular p-aminobenzoic acid, which is the aromatic moiety of the polyene, macrolide, antifungal antibiotic candicidin (Martin and Demain, 1978). Both p-aminobenzoic acid and tryptophan are derived &om chorismic acid. Anthranilate synthetase and p-aminobenzoic acid synthetase may share a protein subunit, and regulation of the synthesis of the common subunit could be affected by tryptophan. Production of aurodox in Streptomyces goldiniensis is controlled by feedback inhibition (Unowsky and Hoppe, 1978; Liu et al., 1979). High yields of aurodox were obtained by reversion of a nonproducer followed by selection of mutants resistant to aurodox. Reversion of nonproducer to aurodox production could have altered the first biosynthetic enzyme of the branch pathway, resulting in a protein desensitized to feedback inhibition. First enzymes of the branch pathways are known to be involved in regulation of metabolic routes (Crawford and Stauffer, 1980). Resistant strains of S . goldiniensis able to grow on 2 gm/liter of aurodox produced higher than 2.5 gmAiter of aurodox. These resistant strains were further improved for antibiotic production by mutagenesis. Strains so

76

VEDPAL SINGH MALIK

selected maintained aurodox resistance even when passed many times in the absence of antibiotics. In these mutant strains, resistance to aurodox could have become constitutive as compared to the inducible aurodox resistance of the parent culture, and constitutive aurodox iesistance may partially account for the increased antibiotic production. The speed with which aurodox inhibits its own synthesis suggests allosteric inhibition of a biosynthetic enzyme rather than repression of enzyme synthesis, the likely mechanism of aurodox regulation. Other reports of feedback inhibition and end product inhibition have appeared in the literature. Agroclavine and elymoclavine inhibit dimethylallyltryptophan synthetase. Anthranilate synthase is feedback-inhibited by elymoclavine (Floss et al., 1974). In protoplasts of Cluuiceps, elymoclavine (0.2 m M ) inhibits the incorporation of tryptophan into the elymoclavine. The end product (gramicidin S) adversely affects the activity and stability of the gramicidin S synthetases from Bacillus ln-evis. End product inhibition is very frequent, but end product destabilization has rarely been reported. Two serine racemases have been partially purified from the Dcycloserine-producer Streptomyces garyphalus. One of them is not inhibited by D-cycloserine. Cycloheximide inhibits its own synthesis when added to producing cultures of S. noursei (Spizek et al., 1965)or S . griseus (Kominek, 1975). Accumulation of penicillin by P. chrysogenuin is inhibited by supplements of penicillin to the growing culture (Gordee and Day, 1972) but the mechanism of inhibition by penicillin of its own biosynthesis is not known. Sulfur metabolism also influences the yields of p-lactam antibiotics. Sulfur for penicillin biosynthesis can be derived from sulfate via the sulfate reduction pathway and from methionine via reverse transsulfuration (Drew and Demain, 1977). Hihg-penicillin-yielding mutants like 4176 take up inore sulfur from the medium than the wild type (Tadrew and Johnson, 1958). Blocked mutants of C. acreinoniuin demonstrate the difference in sulfur metabolism between P. chrysogenum and C . ucremoniuin (Treichler, et al. 1979). Anabolic synthesis of cysteine occurs as a result of the reaction of O-acetylserine with sulfide in the presence of O-acetylserine sulfhydrylase. Cysteine is also synthesized by an alternative route of sulfide fixation in the presence of O-acetylhomoserine sulfhydrylase (methionine synthase). This yields homocysteine, which is transposed to cysteine by the catabolic transsulfuration enzymes, cystathionine P-synthase and cystathionine y-lyase. Both pathways influence antibiotic yield. A mutant of C. acwmonium was boosted in L-serine sulfhydrylase level and utilized an increasing amount of sulfate for cephalosporin biosynthesis. The mutant was sensitive to methionine because it maintained a large pool of cysteine (Komatsu and Kodaira, 1977).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

77

D. CATABOLITE REPRESSION The term “catabolite repression” has been used to explain effects of rapidly utilizable energy sources on the expression of dispensable catabolic pathways. The mechanism of catabolite repression has been exhaustively investigated with the lac operon of E. coli. Lac operon in E . coli is not derepressed by inducers such as lactose in the presence of readily utilizable sugars such as glucose. Besides control of the lac operon by repressor, catabolite repression exerts an additional overriding control. Exhaustion of glucose or rapidly metabolizable carbon source results in slow growth rate and increased intracellular level of cyclic 3’,5’-adenosine monophosphate. This nucleotide is an effector molecule, which together with a catabolite activator protein (CAP) controls the lac operon positively by binding to the operator and making it available for transcription, provided repressor is removed by inducer. What controls CAMP levels under a variety of growth rates is not known. Catabolite repression is a phenomenon where certain nonessential enzymes, which may be necessary only in a given environment at a particular time, are repressed by catabolic products of a readily utilizable carbon source. Whatever the nature of mechanisms, they are probably operative in delaying and reducing antibiotic production by a rapidly used carbon source such as glucose. Production of many antibiotics is inhibited when glucose is used as a carbon source. However, this does not show that the inhibitory effect of glucose on antibiotic synthesis is attributable to catabolic repression, as in the case inhibition ofp-galactosidase in E . coli (Magasanik et al., 1974). The negative effect of glucose has been observed on the yield of penicillin (Johnson, 1952), actinomycin (Gallo and Katz, 1972), streptomycin (Demain and Inamine, 1970), indolmycin (Hurley and Bialek, 1974), kanamycin (Satoh et al., 1976), and puromycin (Redshaw et al., 1979). 0-Demethyl puromycin 0-methyltransferase, which catalyzes the final step in puromycin biosynthesis, is repressed by glucose (Sankaran and Polgeli, 1975). However, the effect of glucose on antibiotic synthesis is not universal. Most aminoglycosides are derived from glucose and their synthesis is not repressed by glucose. Chloramphenicol synthesis is not repressed on glucose and depends on the nutrient combination of the growth medium. Neither glucose nor phosphate represses chloramphenicol production (Malik, 1972). Aharonowitz and Demain (1978) examined the cephalosporin production by S . clavuligwus. Growth on preferred carbon sources such as glycerol and maltose produced high biomass but the specific production of cephalosporins

78

VEDPAL SINGH MALIK

decreased as carbohydrate concentration was increased. Maximum cephalosporin yields were not obtained under conditions supporting highest biomass production. Poorer carbon sources such as starch, a-ketoglutarate, and succinate yielded more cephalosporin, and antibiotic production was closely associated with growth. The negative effect of glucose on bacitracin production was also first thought to be attributable to catabolite repression, but later proved to be attributable to acid production and lowered pH (Haavik, 1974). Coumermycin A can be produced in fermentation broths that comprise a wide variety of assimilable sources of carbon and nitrogen. Under certain conditions, glucose or ammonium salts do not suppress the biosynthesis of antibiotic. Improved yields result from the addition of phosphates and chlorides (Godfrey and Price, 1972). Phenoxazinone s ynthetase catalyzes the synthesis of actinocin, the chromophore of the antibiotic actinomycin. The de nouo synthesis of this enzyme occurs late in the growth cycle when glucose has been exhausted from the medium. In general, those carbon sources that support vigorous cellular growth are most effective in suppressing actinomycin synthesis. The repressive effect of glucose is insignificant once actinomycin synthesis has been well established. Several sugars, acetate, citrate, and pyruvate repress phenoxazinone synthetase formation. This transient inhibition of actinomycin synthesis once antibiotic production has begun may not only be attributable to catabolite repression as it operates in control of lac operon of E . coli but may also involve glutamine synthetase and nitrogen metabolism (Katz, 1967, 1968). Industrial microbiologists have solved the problem of catabolite repression of antibiotic production by medium manipulation. One way is to use two carbon sources in the fermentation. Low concentration of readily utilizable carbon source allows good growth of the organism. A second, nonrepressive, slowly utilizable carbon source is metabolized during antibiotic production. Another way is the continuous feeding of the repressive carbon source at such a slow rate that the inhibitory effect is not exerted. In fact, today most penicillin is no longer made with lactose (Salter0 and Johnson, 1953) but is made with continuous slow feeding of low levels of glucose (Demain, 1968). The p-lactam antibiotic produced in S. clauuligerus (Ahronowitz and Demain, 1978) and C . acremoniunz (Mehta et al., 1980) is limited by glycerol. The inhibitory efyect of glycerol may be attributable to altered membrane structure and permeability and not attributable to catabolite repression. The macrolide chroinophore of polyenes is synthesized from acetate and propionate via the polyketide pathway. The aminosugar moieties found in polyene macrolides are derived from glucose. Glucose is the preferred carbon source for the production of the polyene antibiotic candicidin (Martin

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

79

and McDaniel, 1975). However, in pilot fermentations, high initial concentrations of glucose retarded growth and produced abnormal fermentation patterns. Slow feeding of glucose as in the penicillin fermentation (Pirt, 1976) increased the yield of the candicidin and candihexin antibiotics. Increase in yield was similar when glucose level was maintained at 5 or 15 gm/liter. Respiration rate was higher when glucose was slowly fed, with most polyene remaining attached to the producing cell. Maximal growth rates and final cell mass accumulations were lower, but glucose utilization rates were higher in fermentations where glucose feeding was slow. Tereshin (1976) increased the rate of nystatin production several fold by carbohydrate feeding of various ages of the producing organism. Tereshin (1976)found that glucose and mannose support identical growth and candicidin production in a synthetic medium. Galactose, fructose, arabinose, maltose, sucrose, and lactose decreased candicidin yield. Disaccharides were also poor carbon sources for growth and mycoheptin production by Streptoverticillium mycoheptinicum. Starch and glucose proved to be the best carbon sources for mycoheptin production. Specific antibiotic production was lower using starch as a single carbohydrate source, but biomass yield was higher with starch than with glucose. Lower alcohols (methanol, ethanol, propanol) stimulate polyene production. Intermittent addition of glucose to fermentation increased the yield of amphotericin B and heptaene. Isolation of constitutive mutants for secondary metabolite production is difficult because of the fact that precursors also have to accumulate above a certain level even if growth-linked repression is lifted. However, it should be possible to obtain mutants resistant to catabolite repression. As a matter of fact, the report by Light that a mutant of Penicillium patulum can be obtained that produces 6-methylsalicylic acid synthetase during growth rather than after growth is encouraging. Certain interconverting enzymes of antibiotic synthesis are repressed by glucose, such as mannosidostreptomycinase or mannosidase, which converts undesirable mannosidostreptomycin to streptomycin (Demain and Inamine,

1970). Ragan and Vining (1978) have tested the general hypothesis that overall control of secondary metabolism is mediated by catabolite repression. They measured CAMPlevels in a culture of S . griseus that produced streptomycin during stationary phase of growth (Cella and Vining, 1975). Concentrations of CAMP were highest during active growth when no streptomycin was being produced. The level of CAMP had declined 90% by 5 hr before streptomycin production was initiated. The low concentration of CAMP was found during streptomycin accumulation. These results of Ragan and Vining (1978) suggest that a catabolite repression-type mechanism correlated with an in-

80

VEDPAL SINGH MALIK

crease in the intracellular cyclic 3',5'-adenosine monophosphate concentration does not directly mediate onset of streptomycin biosynthesis in S. griseus. In E . coli, catabolite repression controls inducible catabolic pathways. Secondary metabolism is not a catabolic, but a biosynthetic process. Time required for catabolic enzyme production after peak CAMP levels are attained is about 3 min. However, streptomycin appears in the medium 1 hr after the increase in the specific activity of a biosynthetic transaminase (Horner, 1964). The delay in the appearance of streptomycin in the medium is probably attributable to the lack of accumulation of precursors from which streptomycin is produced. Endogenous accumulation of starting precursors and derepression (induction) of enzymes responsible for the synthesis of secondary metabolites may have to coincide for onset of secondary metabolism. When endogenous levels of starting precursors begin to decrease, synthetases involved in secondary metabolism may start to decay and the rate of accumulation of secondary metabolites in the medium starts to decrease. The experiments of Ragan and Vining (1978) show that streptomycin accumulation in the medium begins 5.5 hr after the peak intracellular CAMP concentration in S. griseus. These studies suggest that the CAMPlevels and streptomycin production are not directly linked but do not rule out an indirect cascade mechanism mediated by effectors. Involvement of CAMP in turimycin production by Streptomyces hygroscopicus has been suggested by Gersch et al. (1978).These authors recorded B decrease in the concentrations of both CAMP and cyclic 3',5'-guanosine monophosphate at the start of turimycin biosynthesis. The intracellular CAMP concentration was claimed to be directly correlated with the amount of growth and inversely correlated with turimycin production. However, intracellular CAMP and cGMP concentration increased when turimycin production was discontinued (Gersch, 1980). Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'triphosphate 3'diphosphate (pppGpp) are important pleiotropic signal molecules in a control system that senses an amino acid deficiency and redirects various cellular activities in response (Lagosky and Chang, 1980). In E . coli, they are synthesized by the ATP:GTP pyrophosphate transferase; stringent factor protein (relA gene product) on ribosomes as a result of the binding of a codon specific for uncharged tRNA during amino acid starvation. In B . suhtilis and E . coli, their intracellular level increases during amino acid starvation and carbon stepdown. Because of the importance of the these nucleoside oligophosphates in metabolic regulation in bacteria, it is possible that changes in their intracellular concentration may be correlated with the onset of secondary

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

81

metabolism. With this in mind, An and Vining (1978)measured ppGpp and pppGpp in S. grieseus during logarithmic and stationary growth phase. They concluded that initiation of streptomycin production in S . griseus is not directly controlled by ppGpp. During exponential growth of S . griseus, the levels of ppGpp and pppGpp were several fold higher than in the stationary phase when antibiotic was produced. Levels of these nucleotides decreased sharply when the culture entered stationary phase. Production of streptomycin started several hours after concentrations of ppGpp and pppGpp had fallen. High ppGpp level does not inhibit streptomycin production, because streptomycin production is not delayed in a glucose starvation medium despite a rising ppGpp concentration. Ragan and Vining (1978) suggest that depletion of phosphate from the medium may be the cause of the sharp decrease in intracellular cAMP concentration as S . griseus enters stationary phase. Exhaustion of phosphate is a prerequisite for initiation of streptomycin biosynthesis, and the decrease in cAMP levels may just be a response to the metabolic switching, rather than a cause of initiation of streptomycin production.

E . ENZYMEMODIFICATION Irreversible inactivation of specific enzymes can regulate flow of precursors via competing metabolic pathways. In vivo degradation and covalent modification of enzymes can diminish wasteful cycling of metabolites through unproductive pathways (Switzer, 1977). Some examples are phosphorylation of pyruvate dehydrogenase in Neurospora crassa (Wieland et al., 1972); the irreversible dissociation of glutamine synthetase induced by NH4+ions in Candida utilis (Sims et al., 1974);the adenylation of glutamine synthetase in Streptomyces and in E . coli (Tyler, 1978; Streicher and Tyler, 1980); the deacylation of citrate lyase in Rhodopseudomonas gelatinosa (Giffhorn and Goltschalk, 1975); the oxidation of an iron-sulfur center of glutamine phosphoribosylpyrophosphate amidotransferase in B . subtilis (Turnbough and Switzer, 1975); and the proteolysis of uridine nucleosidase in yeast (Magni et a l . , 1978; Holzer and Heinrich, 1980; Laskowski and Kato, 1980). Besides an enzyme being inhibited or inactivated, it can be modified (glutamine synthetase), or altered (RNA polymerase), or degraded. Regulation of the quantitative aspects of enzyme production may present a problem. Concentrations of cellular enzymes are regulated so that neither too much nor too little is produced by normal cells. It has not been proved that the “correct” amount of enzymes are made by cells engaged in secondary metabolism.

82

VEDPAL SINGH MALIK

F. FACTORS Mutants of S. griseus that produce little streptomycin and do not sporulate regain the sporulation and antibiotic production if a small quantity of broth of the parent culture is added. The active substance in broth, called factor A, is 2s-isocapryl-oyl-3R-oxymethyl 6-butyrolactone (Kleiner et d., 1976). All industrial strains of S. griseus used for streptomycin production make factor A, traces of which stimulate sporulation, transamiriating enzymes, and streptomycin production up to a level of grams per liter. The molecular formula for factor A is CI:3H'1.t04.All S. griseus strains require factor A for expression of their normal life cycle. The factor A is also produced by Streptoinyces bikiniensis, which produces streptomycin. However, as streptomycin-producer Streptoinyces alhus does not produce factor A, it is not always required for streptomycin production. Only traces of it are needed for a thousand-fold stimulation of antibiotic synthesis; therefore, its role is not of a precursor (Ensing, 1978). The mutants deficient in factor A production bind factor A. Factor A activates an enzyme that hydrolyzes NADP. As a result of the splitting of NADP, adenosine diphosphoribose phosphate (a specific inhibitor of glucose-6-phosphate dehydrogenase) accumulates, resulting in the altered pattern of glucose metabolism in a streptom ycin-producing organism. Another protein, called factor C, is produced late in the growth phase and stimulates sporulation in S. griseus. This cytodifferentiation factor stimulates RNA synthesis and reverses the inhibition by actinomycin D of RNA and protein synthesis in E . coli, B . subtilis, and S. griseus. Factor C raises the T , of the DNA and affects its structure in such a way that mRNA production from latent cell differentiation genes is increased. The characterization of factor C and its mode of action could provide valuable information on the regulation of sporulation in streptomycetes. Some terpene-like extractable molecule effects development of S. alboniger (Pogell, 1975). G. GLUTAMINESYNTHETASE Glutamine is a donor of amino groups in the biosynthesis of all amino acids, the purines, pyrimidine nucleotides, and complex carbohydrates. This central role of glutamine in cellular metabolism is in keeping with the diversity and flexibility in the allosteric control of glutamine synthetase activity (Magasanik, et al., 1974; Tyler, 1978). Glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), E C 6.3.1.2) catalyzes the synthesis of glutamine from glutamic acid and ammonia.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

glutamate

83

+ NH3 + ATP + glutainine + ADP + Pi

In E . coli and other enteric bacteria, the synthesis of glutamine is regulated at two levels:

1. The level of transcription of the glnA gene, which is inversely proportional to the availability of nitrogen in the medium, regulates the amount of glutamine synthetase protein in the cells (Tyler, 1978). 2. Covalent modification of glutamine synthetase protein controls the activity of glutamine synthetase to synthesize glutamine (Prusiner and Stadtman, 1973) The addition or removal of an AMP moiety inactivates or activates the enzyme. Increase in the level of glutamine synthetase resulting from ammonia limitation is responsible for the activation of synthesis of enzymes that supply the cell with ammonia and glutamate. Ammonia limitation in cells growing with glucose as a source of carbon results in the increase in the intracellular ratio of 2-ketoglutarate to glutamine, stimulating deadenylylation and consequent derepression of glutamine synthetase. This shift is ultimately responsible for glutamine synthetase-mediated induction of enzymes that produce ammonia and glutamate from other sources. When nitrogen is in excess, covalent modification by adenylylation of E . coli glutamine synthetase converts this enzyme to a less active form. Adenylylation is catalyzed by the enzyme glutamine synthetase adenyltransferase and protein PII. The protein PII is also covalently modified by uridylation, catalyzed by uridyl transferase. In 1974, Boris Magasanik and his collaborators, working with Klebsiella aerogenes, noted that mutant strains GlnB (PI,)and GlnD (uridyl transferase) possessed little glutamine synthetase that was highly adenylated. On the other hand, GlnE (adenyl transferase) mutants contain elevated levels of glutamine synthetase that was not adenylated. Based on these results, it was suggested that glutamine synthetase controls transcription of its own structural gene (glnA),and that adenylation of glutamine synthetase modifies it as a regulator of transcription. The results of Garcia et al. (1977) threw a monkey wrench in the hypothesis of Magasanik when they reported that the product of a newly identified gene (glnF)is involved in the synthesis of glutamine synthetase in Salmonella and probably also in E . coli. GlnF is located far away from glnA and glnD genes of Salmonella and does not map in the region of the Salmonella chromosome that corresponds with GlnB (PII)and glnE (adenyl transferase) genes of Klebsiella. GlnF extracts contain normal amounts of all

84

VEDPAL SINGH MALIK

proteins involved in covalent modification of glutamine synthetase and glnF may be involved in the regulation of nitrogen metabolism. As a matter of fact, most glutamine synthetase preparations used by co-workers of Magasanik were not absolutely pure and might have contained glnF product as a minor contaminant. If glnF product is indeed a regulatory protein, then only a few molecules per cell would be enough to exert the regulatory effect. In Gram-positive bacilli, the mechanism or regulation of nitrogen metabolism has not been well explored. Covalent modification of glutamine synthetase in B. subtilis (Deuel et al., 1970)and Bacillus stearotherrnophilis (Hachimori et al., 1974; Wedler and Hoffman, 1974) does not occur. However, Streicher and Tyler (1980) have demonstrated that the activity of glutamine synthetase in a Gram-positive, filamentous, spore-forming bacterium Streptomyces cattleya (Kahan et al., 1979)is regulated through covalent modification, as in enteric bacteria. S. cattleya produces a p-lactam antibiotic thienamycin. Radiolabeling experiments of Streicher and Tyler (1980) demonstrate that addition of ammonium chloride to S. cattleya cells growing under nitrogen limitation conditions leads to rapid adenylylation and inactivation of glutamine synthetase. As in E . coli, the adenylylation reaction in S. cattleya crude extracts required ATP and was stimulated by glutamine and inhibited by a-ketoglutarate; the ratio of these two metabolites regulated the adenylylation state of glutamine synthetase. Tronick et al. (1973) have reported that glutamine synthetase of Streptoinyces rutgwsenis and Streptomyces diastatochromogenes is not adenylated. However, when S. cattleya was grown under conditions of Tronick et al. (1973), the glutamine synthetase was present in a very low adenylylation state and escaped detection by the methods used. The glutamine synthetase of these two streptomycetes is probably adenylylated and should be further examined by the methodology and growth conditions used by Streicher and Tyler (1980). Aharonowitz (1979, 1980) found that the level of glutamine synthetase in the S. clavuligerus cells responds to the source of nitrogen in the growth medium. Streicher and Tyler (1980) found more than a 20-fold increase in glutamine synthetase protein levels in derepressed cultures of S. cattleyu. The increased glutamine synthetase protein levels in depressed S. cattleya cells could be attributable to an increased rate of transcription of the S . cattlqa glnA gene or to decreased rates of degradation of glutamine synthetase protein or gZnA mRNA. The role of glutamine synthetase in the control of the onset of thienamycin production in S. cattleyu is not known, but definitely deserves consideration. In the presence of ammonia, the active octameric glutamine synthetase of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

85

the yeast Candida utilis is converted to the less active tetramers and then into inactive monomers (Sims et al., 1974). Sanchez et a2. (1980) suggest that glutamine rather than glutamate is the amino donor for the synthesis of amino acids that are involved in penicillin synthesis. High concentrations of ammonium ion prevents glutamine synthetase formation and thereby results in a decreased intracellular glutamine pool. During penicillin production, irreversible transamination catalyzed by glutamine transaminase could produce penicillin precursors L-cysteine, L-valine, and L-a-aminoadipic acid from the a-keto acids glyoxylate, a-ketoisovalerate, and a-ketoadipate, respectively. Leucomycin production by Streptomyces kitasatoesis is inhibited by high concentration of ammonium ion but not so much by inorganic phosphate. Addition of 0.5 to 2%water-insoluble magnesium phosphate to the leucomycin production medium stimulated leucomycin production. Omura et al. (1979) claim that magnesium phosphate stimulates conversion of media glycine into L-serine. These authors further speculate that magnesium phosphate stimulates leucomycin production by trapping free ammonia from the media. H. ENERGYCHARGE The biosynthesis of excessive amounts of secondary metabolite may be energetically favorable for the utilization of metabolites accumulated as a result of a faulty regulation of primary metabolism. The so-called energy charge has been used to evaluate the intensity of energy metabolism and its regulatory role. This parameter, defined as (ATP) (MADP)(ATP) (AMP) reflects the energy state of the cell. Phosphate (0.3-300mM) supports extensive microbial growth but a concentration of inorganic phosphate of 10 m M and above exerts a depressive effect on the synthesis of many secondary metabolites belonging to different biosynthetic groups. Many antibiotics are industrially produced at growthlimiting concentrations of phosphate (Martin, 197%). Phosphate addition inhibits antibiotic synthesis by stimulating growth of the nongrowing candicidin-producing S . griseus. Accompanying the inhibition was a rapid increase in intracehdar ATP concentration (Martin et al., 1979~).A rapid decrease in intracellular ATP could be associated with the onset of antibiotic synthesis. The intracellular level of all adenylates in the wild-type isolate was 10-fold higher compared to that in the chlortetracycline production strain. The production culture was suppressed overall in adenylate synthesis. However, the values of the energy charge were similar in the production and the

+

+

86

VEDPAL SINGH MALIK

wild-type cultures. The decrease during antibiotic production may not be attributable to a low level of ATP but may be the result of an increase in ADP and AMP. The signifcant rise in the A M P level in the wild-type strain may reflect increased AMP synthesis. The rise in the AMP level could also be attributable to the splitting of diphosphate bonds in ATP and ADP. The phosphatase increased sharply during chlortetracycline production although ATP was minimal. This nonspecific phosphatase, which splits all diphosphates is repressed by inorganic phosphate and probably generates phosphate during phosphate limitation while metabolism shifts from the adenylate phosphorylation system to the polyphosphate phosphorylation system. With increased concentration of inorganic phosphate in growth medium, the ATP concentration is maintained at a high level throughout the cultivation, and the energy charge values are considerably lower than under phosphatelimiting conditions. Cell growth is favored, and cellular polyphosphates increase. Phosphatase enzymes involved in antibiotic synthesis are repressed. The level of anhydrotetracycline hydratase, an inducible enzyme of the tetracycline biosynthetic pathway, catalyzing the hydration of anhydrotetracycline to 5a, 1la-dihydrotetracycline, is sharply diminished on increasing orthophosphate concentration above the optimum value. Starting precursors of antibiotic synthesis in S . aureofaciens could be synthesized by specific pathways different from those yielding the same intermediates for growth. Intensity of antibiotic production occurs under conditions not the best for growth. Besides synthesis of enzymes of antibiotic biosynthesis, unfavorable growth environment induces other metabolic changes, e.g., the activation of regulation mechanisms governing Pi regeneration. The process represents an overall economizing rearrangement of the culture metabolism immediately after growth termination.

VII. Regulation of Autotoxicity Many secondary metabolites have no biological toxicity against the organism that synthesizes them because they lack a target site and can, therefore, be exempted from the penality of toxicity. On the other hand, many prokaryotes produce autoinhibitors and must have mechanisms of selfdefense against their own autotoxic metabolites (Lisivinova et a l . , 1979). Various mechanisms of antibiotic tolerance in producer organisms have been extensively reviewed (Demain, 1974b; Vining, 1979; Malik, 1979b) and will not be discussed here. The ability of an organism to produce an autotoxic metabolite can be curtailed by the level of sensitivity of the producer to the autoinhibitor that it produces. In many cases, resistance is inducible and does not even require

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

87

the synthesis of the autotoxic metabolite. For example, chloramphenicol resistance in S . venezuelae is induced by externally added chloramphenicol under conditions when no chloramphenicol is produced (Malik, 1979b).This suggests that resistance and synthesis of chloramphenicol in S . venezuelae are independently controlled and may even constitute separate operons. The first few molecules of endogenously produced chloramphenicol also induce resistance. Like many other antibiotic resistance markers, chloramphenicol resistance in the producer S . veizezuelae is very unstable and may be coded on a translocatable element. The close coupling between the regulation of biosynthesis and development of resistance to autoinhibitors could be of evolutionary significance. However, no general rules can be formulated to predict the location of genes involved in resistance and synthesis of these autoinhibitors. Genetic inapping in s. coelicolor suggests that genes involved in the synthesis of methylenoinycin A and those for resistance are located on plasmids. A. REGULATION OF

PERMEABILITY

During production of antibiotics, a marked development of mesosome-like membranous structure usually occurs. Although mesosoines are known for their important primary metabolic energy-producing processes, membrane-bound areas and vacuoles could be associated with production, accumulation, and release of antibiotics froin the producing cell. This cellular compartmentalization resulting in localization of antibiotics in such areas may help to separate the toxic antibiotic from potentially sensitive sites in the cell. If they accumulate in veiscles formed by invaginations of inembrane, they could then be secreted through cell walls and never reenter the cell. Enzymes catalyzing terminal steps of antibiotic synthesis could be associated with membranes as in many other microbiol enzymes. Aurodox inhibits both protein synthesis and the release of the elongation factor Tu from the ribosomes of the producing organism S . goldiniensis (Unowsky and Hoppe, 1978). The inducible resistance to aurodox in the producer organism is probably attributable to alteration of membrane permeability (Liu et al. , 1979) and similar to development of resistance to tetracycline inhibition of protein biosynthesis. Chloramphenicol resistance in S. venezuehe is not attributable to chlorarnphenicol acetyltransferase but to a permeability barrier. Study of the genetic control of resistance to chloramphenicol in producing streptomycetes offers the opportunity to understand control of membrane permeability in these industrially important organisms. The role of the peptide antibiotic bacitracin in a producer organism has

88

VEDPAL SINGH MALIK

been investigated and the environmental regulation of sporulation, expecially with respect to manganese has been elaborated. The exact mechanism of resistance of the producer to bacitracin remains to be discerned, but the resistance phenotype is dependent on the expression of bacitracin synthetase genes. Bacillus brevis becomes impermeable and resistant to edeine during antibiotic production. Nascent edeine exists in an inactive form in postlogarithmic-phase producer cells; it is bound via a thioester linkage to a fast-sedimenting fraction containing polyenzyines of edeine biosynthesis and D N A membrane complex (Borowska and Szer, 1976). In industrial fermentation, fatty acids and their esters are added to growth media to stimulate tylosin production. Exogenous fatty acids alter the fatty acid composition of cellular phospholipids. Cells grown in the presence of oleic acid show different transport properties for valine. Alteration in permeability could influence the excretion of an antibiotic. The stimulation of neomycin and streptothricin production by exogenous oleic acid is attributable to alteration of membrane permeability (Arima et al., 1973; Okazaki et al., 1974). Even though neomycin is not a polyketide-derived antibiotic, its final yield in fermentations of a S. faradiae mutant that requires oleic acid for neomycin formation depends on the cellular fatty acid spectrum (Atima et al., 1973; Okazaki et d.,1974) The high antibiotic production capability of certain phage-resistant strains could be attributable to their altered membranes (Malik, 197913). Modification of cell membrane may release cellular metabolites so that they are diluted into the production media and do not exert regulatory controls (Demain and Birnbaum, 1968). Polyene-resistant mutants could be altered in cell membrane; they secrete large amounts of desired products (Fisher, 1980). Mutants of Penicillium stolonifmum resistant to amphotericin B, filipin, and nystatin were altered in sterol composition (Wilkerson et al., 1978). Some mutants produced altered amount of ergosterol and unique sterols. One mutant produced increased amounts of ergosterol and mycophenolic acid, a metabolite partially derived from farnesylpyrophosphate. Mutants of C. acremonium or Cephalosporium polyaleuruin resistant to nystatin, kabicidine, or trichomycin secrete more than 10 gm/liter of cephalosporin C. A kabacidine-resistant mutant of Fusurium secretes high yields of ergosterol and of alkaline protease (Suzuki et al., 1974). Another kabicidineresistant mutant of Trichoderma reesei is derepressed in production of cellulase (Gallo, 1979). Study of the structure of the membranes of these hyper-secreting strains could establish the type of membrane alterations that allow metabolite secretion.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

89

B. MODIFICATION OF TARGETSITE Prokaryotic streptomycetes produce many antibiotics that are inhibitors of protein synthesis and act at the level of the prokaryotic 70 S ribosome. Ribosomes from some of the autoinhibitor-producing streptomycetes are modified in such a manner that the autoinhibitor no longer binds to their ribosomes (Vining, 1979). 1. Methylation of Ribose

Resistance of S. azureus to its own polypeptide antibiotic thiostrepton is attributable to methylation of the ribose of its 23 S ribosomal RNA. Whether the methylase responsible for resistance is inducible or constitutive is still unresolved (Cundliff and Thompson, 1979). Methylation of 23 S rRNA occurs before assembly of the 50 S ribosomal subunit is completed. Thiostrepton-resistant methylase of S. nzureus is an RNA-ribose methylase that introduces a single methyl group into adenosine in 23 S rRNA. Ribose methylation is a novel mechanism of antibiotic resitance and high frequency of eukaryotic rRNA methylation may determine resistance to thiostrepton.

2. Methylation of Adenosine Base Two other examples involve base methylation as a mechanism of posttranscriptional modification of rRNA. 1. Kasugamycin-resistant mutants of E . coli possess undermethylated 16 S rRNA and lacks N6,N6-dimethyladenosine normally located near the 3’ terminus (Helser et d., 1971). 2. Overmethylated 23 S rRNA is responsible for the so-called MLS phenotype that is found in Staphylococcus aureus, Streptococcus, and Streptomyces erythreus (Weisblum and Graham, 1979; Horinouchi and Weisblum, 1980).

The presence of N6,N6-dimethyladenosine in the 23 S rRNA of S . aureus is responsible for coresistance to macrolide, lincosamide, and streptogramin B-type antibiotics (MLS resistance) (Weisblum and co-workers). MLS resistance in S. aureus and Streptococcus pyogenes is encoded on plasmids. In fact, a fragment of plasmid DNA from S. pyogenes that codes for the 23 S rRNA methylase cross-hybridizes with the plasmid coding for MLS resistance in S. aureus (Weisblum et al., 1979). Antibiotic resistance genes could have originated in the producing organism (streptomycetes, in most cases) and somehow were transferred to the

90

VEDPAL SINGH MALIK

true bacteria (Benveniste and Davies, 1973). Graham and Weisblum (1979) showed that an erythromycin-producing strain, S. erythreus NRRL 2338, has the same MLS resistance phenotype as that observed in S. aureus and S. pyogenes. This culture contained N N ‘-dimethyladenosine in its 23 S rRNA, which was not the case in several other species of streptomycetes, including some other macrolide producers (Cocito, 1979). Their studies, however, did not suggest the existence of any extrachromosomal DNA in S. mythreus. By methylating its 23 S rRNA, S. erythreus becomes resistant to macrolide-lincosamide-streptogramin B(MLS)-type antibiotics. Horinouchi and Weisblum (1980) have cloned the inducible erythromycin resistance gene from S. uureus into B. suhtilis. The cloned 1442-base pair TAQ1A fragment codes for a leader peptide (19 amino acids) and a “29-K protein” that consists of 243 amino acids. The promoter region for 29-K protein has 4 complementary inverted repeat sequences named “1, 2, 3, and 4.” The C-terminal half of the leader peptide is coded by sequence 1, which is complementary to 2. Sequence 2 is complementary to 3, and sequence 3 is complementary to 4. Ribosome binding site for the synthesis of 29-K protein lies in a loop formed by the complementarity of sequence 3 4. Like attenuators (Keller and Calvo, 1979; Rosenberg and Court, 1980; Johnston et al., 1980), the promoter of 29-K protein does not have transcription termination signals. Ribosomes engaged in leader peptide synthesis could be partially inhibited b y optimal inducing levels of erythromycin, resulting in accumulation of partially blocked ribosomes in sequence 1. Horinouchi and Weisblum (1980) postulate that accumulation of a high level of stalled ribosomes in sequence 1can perturb the translationally inactive double-stranded inverted repeats between sequences 1 and 2 and sequences 3 and 4, resulting in hydrogen binding of sequence 2 with 3. This results in induction of erythromycin resistance, because the ribosome binding site for the synthesis of 29-K protein, which was otherwise not available because of the association of sequence 3 with 4, is free. Cerulenin resistance in a cerulenin-producing fungus is attributable to a cerulenin-insensitive fatty acid synthetase (Kawaguchi et al., 1979).

‘,

+

c. BIOTRANSFORMATIONAND REGULATION OF

CATABOLISM

Most organisms can also inactivate the produced secondary metabolite. Mechanisms that control antibiotic resistance and inactivation in the producing organism have commercial significance because their manipulation is used to increase the yield of antibiotics in industrial fermentations. Enzymes that convert antibiotics into biologically inactive compounds in-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

91

fluence the final yield of antibiotic accumulated in the fermentation broth. For instance, lincomycin is oxidized to lincomycin sulfoxide by an oxidoreductase of Streptomyces lincolnensis (Argoudelis and Mason, 1969). Polymixin E is decomposed by a protease produced by Bacillus polymyxa, which synthesizes this antibiotic (Woyczikowska et al., 1973). Chloramphenicol is degraded by the producing streptomycetes, which contain the enzyme chloramphenicol hydrolase (Malik and Vining, 1970). The mikamycin-producer Streptomyces mitakaensis contains the enzyme mikamycin P-lactonase, which causes low titers of the produced antibiotic in the medium. A kinase from streptom ycin-producing S. griseus will phosphorylate streptomycin at the 3 position of the N-methyl-L-glucosamine ring. Inactivation of the streptomycin by phosphorylation is controlled by the level of phosphate in the medium. The kinase may be responsible for the resistance of the producing organism to streptomycin. A second phosphorylating enzyme (ATP:dihydrostreptomycin-6-P-3a-phosphotransferase) phosphorylates the 3a’ position of the dihydrostreptose moiety of dihydrostreptomycin. A third enzyme, streptomycin-6-kinase (ATP:streptomycin-6-phosphotransferase), is found in Pseudomonas aeruginosa (Kida et al., 1975) and S. bikiniensis (Miller and Walker, 1969). Recently, Piwowarski and Shaw (1979) have suggested that resistance to streptomycin in S. bikiniensis may be the result of phosphorylation of streptomycin at the 6 position of the streptidine subunit by a plasmid-borne kinase. This enzyme is not present in logarithmic phase cells, which are not yet committed to streptomycin production and are susceptible to 25 pglml of streptomycin. The kinase activity appears in stationary phase cells 12 hr before streptomycin is detected in the medium. Stationary phase cells are producing streptomycin and are resistant to more than 200 pglml of streptomycin. Certain isolates of S. bikiniensis that have lost the ability to produce streptomycin were selected after treating the culture with acriflavine or ethidium bromide. These cultures were inhibited by 10 pg/ml of streptomycin throughout their growth, did not inactivate streptomycin, and lacked streptomycin-8kinase. These results suggest that phosphorylation by streptomycin-6-kinase plays an important role in resistance to streptomycin in S. bikiniensis. The streptomycin-6-kinase could be so located on plasma membrane that it inactivates the Streptomycin that is trying to enter the cell via the polyamine transport system (Holtje, 1978, 1979). Cellular phosphatase may convert the inactive phosphorylated antibiotic into streptomycin before its secretion from the cell. As a result, streptomycin accumulates in the medium and not the phosphorylated derivative. Phosphate inhibits formation of alkaline phosphatases required for the synthesis of streptomycin

92

VEDPAL SINGH MALIK

(Miller and Walker, 1970b), viomycin (Pass and Bojanowska, 1969b), and vancoinycin (Mertz and Doolin, 1973). Dephosphorylation is the final step in the biosynthesis of these antibiotics. In the presence of phosphate, inactive phosphorylated derivatives of streptomycin and neomycin accumulate in the media (Miller and Walker, 1970b; Majumdar and Majumdar, 1970). Streptomyces glaucescens produces hydroxystreptomycin, tetracenomycins, an a-amylase inhibitor, and a bacteriocin. It is also resistant to streptomycin (1 pg/ml) and secretes melanin. In S. glaucescens, genetic determinants for streptomycin resistance, secretion of melanin and chitinase, synthesis of laminarinase, and production of spores are highly unstable (Suter et al., 1978). A frequency of spontaneous mutant phenotypes of up to 1% can be obtained for any of these markers. The mutation frequency can be increased to more than 10% by prolonged cold storage or by cultivation on medium supplemented with ethidium bromide. Mutants in several of the aforementioned traits occur simultaneously. Genetic analysis shows that such phenotypes are attributable to different independent mutations and are not the pleiotropic effects of single mutations. Mutants resistant to high levels of streptomycin (100pglml) occur infrequently. However, mutations that cause increased sensitivity to streptomycin (<1 pg/ml) are common but unstable (Freeman and Hopwood, 1978). Stable streptomycin-sensitive strains can be obtained by several cloning steps of streak purification. Sensitivity of the mutant strains is due to increased accumulation of streptomycin (Hutter et al., 1979). Genes responsible for streptomycin sensitivity and melanin formation have been mapped on the chromosome. The DNA of the mutant and parent strains were labeled with [3H]thymidine and [14C]thymidine,respectively. After digestion with restriction enzymes BainHI and SalGI; the DNAs were mixed and analyzed by agarose gel electrophoresis. The determination of the 14C:3Hratio in various DNA bands indicated the absence of a 13.5-kilobase DNA fragment from the DNA of a streptomycin-sensitive, melanin-negative mutant. This 13.5-kilobase DNA fragment can now be cloned and used as a probe to hybridize to the various DNA fragments of the parents that have been resolved by electrophoresis. This type of analysis may reveal if streptomycin sensitivity is attributable to the participation of a translocatable and insertion element. Other examples of chromosomal instability are oxytetracycline sensitivity in S. rirnosus (Hopwood, 1978; Borisoglebskaya et al., 1979), chloramphenicol sensitivity in S. coelicnlor (Freeman et at., 1977; Sermonti et at., 1977, 1978, 1980; Micheli, 1980), and chloramphenicol sensitivity in S. uenezuelae (Malik, 1979b). S . coelicolor and chloramphenicol-producing S . venezuelae are resistant to chloramphenicol but do not possess chloramphenicol acetyltransferase (Freeman et al., 1977).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

93

An inhibitor of chloramphenicol acetyltransferase is produced by certain streptomycetes (Miyamura et al., 1979). Many variants of chloramphenicol acetyltransferase have been found in many non-chloramphenicol-producingstreptomycetes (Shaw and Hopwood, 1976) and bacteria (Shaw et al., 1979; Liddel et al., 1978; Shaw, 1979). Chloramphenicol resistance due to chloramphenicol acetyltransferase is determined by a transposable genetic element (Tn9), which consists of two direct repeats of the insertion sequence IS1 flanking a region of 1102 base pairs (Chandler et al., 1979). Transposition ofTn9 results in the duplication of a %base pair sequence of the insertion site. One copy of the %base pair sequence is located at each end of inserted element. Alton and Vapnek (1979) have sequenced the 1102-base pair region between the directly repeated IS 1 sequences encoding chloramphenicol acetyltransferase of E . coli. The gene product ofTn9 is expressed in Saccharomyces cerevisiae when the chloramphenicol-resistance determinant is inserted into a 2-pm yeast plasmid. Expression of the bacterial chloramphenicol acetyltransferase gene also occurs in streptomycetes when ligated to a streptomycetes promoter (Bibb et al., 1980b). With the available methodology, it should now be possible to clone and sequence the genetic determinants of chloramphenicol acetyltransferase of streptomycetes. If streptomycetes chloramphenicol acetyltransferase is encoded by a transposable genetic element similar to Tn9 of E . coli, then it could be useful for studying mutagenesis and genetics in streptomycetes. A gene coding for neomycin and butirosin phosphotransferase from a butirosin-producing Bacillus circulans has been cloned and expressed in E . coli (Davies et al., 1979). Many aminoglycoside-modifying enzymes have been described in both producing organisms and in plasmid-carrying bacteria (Dowding, 1979b). Many P-lactamases of Gram-positive bacteria (Imsande, 1978) and penicillin-biiiding proteins of p-lactam-producing Streptomyces cacaoi, Streptomyces olivaceus, and S . clavuligerus have been examined (Ogawara and Horikawa, 1980a,b). Another example of antibiotic inactivation by the producing organism is the metabolism of nebramycin by S. tenebrarius (Stark et al., 1980). Nebramycin activity declines in the medium after reaching a peak. However, nebramycin inactivation does not occur if ample energy source is present in the media. The nature of the inactivation product, when known, could be important if nebramycin is degraded to subunits that could be used to prepare analogs of nebramycin. The control of the enzymatic hydrolysis of cephalosporin C to deacetylcephalosporin C occurs in the producing organism. When either glucose, maltose, or sucrose is fed to a C.acremonium mutant, the formation of an

94

VEDPAL SINGH MALIK

extracellular carboxyesterase acting on cephalosporin C is suppressed (Hinnen and Nuesch, 1975). Derepression of cycloheximide degradative enzymes upon exhaustion of glucose from the medium of cycloheximide-producing S . griseus has been suggested by Kominek (1975).

VIII. Secondary Metabolism, Sporulation, and Exoenzyme Formation Synthesis of antibiotics in the stationary growth phase, which in Bacillus and Streptomyces is the phase of cell differentiation and formation of spores, suggested that antibiotic synthesis and sporogenesis might be linked. However, these two processes are not inseparably interrelated; for example, Pseudornonas strains produce antibiotics but do not form spores. Sporulation and secondary metabolite formation are initiated at the end of logarithmic growth when certain nutrients in the growth medium are depleted. The fact that secondary metabolism and sporulation are initiated at the end of growth makes analysis of the specific genes very difficult. Many genes are released from growth-linked repression at the end of the growth phase, and it is difficult to differentiate between these derepressed gene functions and those that are specific for growth, sporulation, or secondary metabolite formation. The genetics of secondary metabolism and sporulation is much more complex than the genetics of growth because the expression of genes involved in these processes is superimposed on the expression of logarithmic phase genes. Identification of any primary products for any of the genes involved in secondary metabolism or sporulation is yet to be achieved. Another inherent difficulty caused by the pleiotropic nature of most mutations affecting secondary metabolism and sporulation makes it difficult to approach this problem by modern methods of molecular genetics. Bacterial sporulation is a time-ordered sequence of biochemical and morphological events resulting in the conversion of a vegetative cell into a spore. At the end of exponential growth when sporulation begins, cells elaborate several proteolytic enzymes and antibiotics (Bose et al., 1979). Certain temporal and genetic relationships exist between these two early molecular events. However, the exact nature of the relationship remains elusive. In B . subtilis, sporulation was initiated by the presence of excess glucose, ammonium ions, and phosphate; by guanine deprivation of a guanine auxotroph; or by the addition of decoyinine, an inhibitor of GMP synthetase. Under these circumstances, ATP generation and substrate synthesis pro-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

95

ceeds by glycolysis, and citric acid cycle enzymes are not required. Glucose repressed the synthesis of inducible enzymes even when guanine deprivation triggered sporulation. However, enzymes typically found during sporulation were the same whether sporulation was induced by nutrient limitation or by deprivation of guanine nucleotide in the presence of excess glucose, phosphate, and ammonium ions (Vasantha and Freese, 1980). Sporulation and secondary metabolism are favored under similar growth conditions. This may be attributable to some regulatory event that triggers both processes. Many best producers of secondary metabolites are asporogenous mutants. Such mutants could be impaired in the events occurring after the regulatory signal that is common to both sporulation and secondary metabolite formation. In this way, precursors and energy would be diverted to secondary metabolism instead of sporulation. The production of extracellular proteases and antibiotics has been intimately linked with the initial stages of sporulation. Sporulation, serine protease, and cephamycin production in Streptomyces lactamdurans is well coordinated (Ginther, 1979). Most protease-less and antibiotic-less mutants are also asporogenous and many result from pleiotropic effects of mutations (Schaeffer, 1969). Extracellular protease is probably not required for sporulation but intracellular protease may convert coat precursor to coat protein and modify and degrade other enzymes. Antibiotic production may be an earlier event than sporulation in cellular differentiation. Most industrial strains that are potent producers of antibiotics sporulate poorly. High productivity of these strains could be attributable to the fact that precursors of sporulation are channeled into antibiotic formation instead. The subunit pattern in the RNA polymerase from sporulating cells is quite different from that in vegetative cells. The subunit of RNA polymerase from cells engaged in secondary metabolism and vegetative growth have not been studied. Modification of RNA polymerase can control the transcription of a number of scattered genes simultaneously, but what controls the modification of RNA polymerase is not known. Knowledge of regulatory mechanisms that control genes coding for RNA polymerase subunits of genes controlling enzymes that modify subunits is needed. To understand the expression of sporulation genes during developmental sequence, Losick (1981) cloned a B . subtilis gene whose transcription is activated at an early stage of spore development. This gene encodes an RNA of about 400 bases and is called the “0.4-kilobase gene.” The transcription of this gene, which maps near the origin of replication of the B . subtilis chromosome is restricted in mutants that are blocked at the early stages of sporulation. A modified form of RNA polymerase that transcribes the 0.4kilobase gene has been isolated from early sporulating cells of B . subtilis.

96

VEDPAL SINGH MALIK

This modified RNA polymerase lacks (T factor but contains a novel polypeptide subunit. This new subunit, which replaces the (T factor of RNA polymerase, has a molecular weight of 37,000 and is known as P3‘. About six genetic loci known as the “Stage 0” genes control initiation of sporulation in B . subtilis. Mutations in Stage 0 genes SpoOA, SpoOB, SpoOE, SpoOF, and SpoOH block sporulation at the first morphological step, Stage 0, and diminish transcription of the 0.4-kilo11ase gene in vivo. Pa?may be the product of a Stage 0 gene because it turiis on early sporulation genes by modifying RNA polymerase. The “0.3 kilobase gene” of Bacillus subtilis is transcribed at an intermediate stage of sporulation. The promotor of this gene is not recognized and transcribed by the Sigma factors isolated from vegetatively growing cells, but is recognized and transcribed by the RNA polymerase activity isolated froin sporulating cells. In order to determine its function, a recombinant DNA plasmid (~1419) has been constructed to introduce an insertion mutation in the “0.3 kilobase gene.” P1419 replicates autonomously in E . coli and confers resistance to tetracycline and chloramphenicol. However, this recombinant plasmid does not replicate autonomously in B . suhtilis. The plasmid does confer resistance to chloramphenicol, however, if by homologous recombination it can integrate into B . suhtilis chromosome. Cloning of a specific B . subtilis gene into p1419 provides the homology required for integrating into that particular gene. A 68-base-pair fragment of the “0.3 kilobase gene’’ was cloned into ~ 1 4 1 9and , the integer was transformed into €3. subtilis. Plasinid integrated cloqe to the chromosomal site of the “0.3kilobase gene.” Phenotypes of these insertion mutants appears to affect cortex formation. These experiments are of great significance since they provide novel strategies for isolating genes, mutating them in vitro, introducing them into the genome by homologous recombination, and then studying the phenotypic effect of the gene on the constructed strain. This approach of reciprocal genetics has immediate application to the study of secondary metabolism and should therefore be pursued. Because PS7is present in both vegetative and sporulating cells, it may direct transcription of many classes of genes. However, other Stage 0 gene products may affect specific participation of P37in sporulation RNA synthesis. In this way, one or more regulatory protein subunits of RNA polymerase that interchange may be involved in control of sporulation. Different forms of RNA polymerase containing distinct regulatory subunits may control sporulation and secondary metabolism. Little is known about the molecular mechanisms in initiating sporulation or antibiotic formation. Pleiotropic mutations at any one of nine distinct genetic loci block sporulation in B . subtilis at early stages without affecting

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

97

vegetative growth (Piggot and Loote, 1976). These pleiotropic mutations scattered throughout the B. subtilis chromosome are also impaired in Stage O functions, such as antibiotic and protease production, competence for transformation, and sensitivity to surface active antibiotics and phage. Trowsdale et al. (1978) examined the ribosomal composition of these pleiotropic Stage 0 mutants and of the same mutants bearing an additional mutation in another locus abrB, which suppresses the pleiotropic mutant phenotype without suppressing the sporulation defect. Suppressed strains were resistant to polymyxin and wild-type antibiotics and produced extracellular proteases. Some of them produced antibiotic and were nonpermissive for two bacteriophages that only replicate in Stage 0 mutants. All mutants have alterations in different ribosomal proteins, suggesting that a group of structural genes specifying several ribosomal proteins are located in the abrB region near the origin of replication on the genetic map of the B . subtilis chromosome. Without allowing sporulation, these ribosomal alterations permit reading of messages that specify proteins concerned wtih proteases, antibiotics, and other early functions in sporulating cells that are masked in Stage 0 mutants. In wild-type sporulating strains, this mRNA may be read when a change takes place on the ribosome early in sporulation. Stage 0 gene products alter the ribosomes so that they translate certain species of message. Early in sporulation, changes take place in ribosomeassociated proteins (Trowsdale et al., 1978).

IX. Role of Secondary Metabolism Genetics and molecular complexity of various secondary metabolites suggests that their synthesis requires specific enzymes. Because they are accumulated in significant amounts, a significant pool of materials of primary metabolites and energy is used in their formation. Secondary metabolism must, therefore, have a function, to prevent its elimination during evolutionary selection. Like other organisms, microbes have evolved over millions of years. Certain microbes, such as E . coli, are metabolically very efficient. Their metabolism is well regulated and various metabolic steps are always so coordinated that they are in step with each other. Whenever concentrations of any cellular products increase above certain levels, efficient organisms switch off corresponding operons and utilize other well-evolved regulatory mechanisms to prevent accumulation of end products and intermediates. Such tightly regulated microbes with balanced metabolic circuits are of little use to industrial microbiologists because they do not overproduce cellular metabolites. However, in many microbes, certain primary metabolic reactions lack

98

VEDPAL SINGH MALIK

partial or complete regulation. As a result, intermediates and end products of primary metabolism are overproduced. Such organisms have evolved with an alternative strategy to well-coordinated cell regulatory mechanisms (Malik, 1980a). They have developed additional pathways that channel these oversynthesized cellular products into secondary metabolites. This has probably occurred in a series of sequential steps.

1. As a result of some faulty cellular regulatory mechanism, cellular products of primary metabolism probably accumulated. 2. Some cellular enzyme with loose cofactor and substrate specificity acted upon the accumulated primary product and converted it to the poor substrate of a second cellular enzyme. In this manner, multistep pathways for converting overproduced metabolites into secondary metabolites have evolved. 3. These accumulated intermediates were probably poor substrates for the cellular enzymes. As a result, genes for these enzymes were tandemly duplicated. At this stage in evolution, one gene was selected for rigid enzymatic specificity and became fixed in the operons participating in primary metabolism. However, a sister gene coded for enzyme with loose specificity and, as a result of chromosomal rearrangement and reorganization, got fixed in the clusters that participate in transformation of accumulated products into secondary metabolites. In this way, such organisms possess, in addition to genes for growth, genes for production of secondary metabolites. Regulation is an essential condition of life. Metabolic regulation is not a late development somehow grafted on to preexisting metabolic sequences; the sequence and their regulation necessarily evolved together, with tnutation and selection acting in most cases on the same molecules in the development of catalytic efficiency and of regulatory response. Hardly any reaction or pathway can be imagined to be adaptive in the absence of regulation. Evolution of every inetabolic sequence has been accompanied by evolution of control characteristics that cause the sequence to respond appropriately to the needs of the organism under a wide range of internal and external conditions. Just as the individual reaction has meaning only in terms of the sequence in which it participates, the sequence is useful only by virtue of its integration into the overall metabolism of the cell or organism. The hnction of regulatory enzymes (secondary metabolite production) is to affect this integration. Complex regulatory responses result from the simultaneous occurrence of numerous regulatory interactions, each relatively simple in principle. Secondary metabolites might have evolved as a viable option to switching

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

99

off metabolic pathways by various control mechanisms and may be beneficial for the organism. In this way, intermediates and end products of the primary metabolism, which would normally accumulate in the cell, are excreted, whereby their regulatory effect is diminished or completely abolished. Some of these secondary metabolites accidentally happen to be biologically active; some are antibacterial and are even toxic to the organism that produces them. Producing organisms have then evolved with mechanisms that protect against the toxic effects of their own metabolites (Demain, 1974a; Davis, 1980; Vining, 1979; Malik, 1972, 1979b). Ensing (1980) suggested that an antibiotic is involved in the maintenance and control of dormancy of spores in Streptomyces viridochromogenes. The antibiotic is present in spores and prevents spore germination by inhibiting Na+,K+-activated ATPase. As a result, dormant spores oxidize glucose but produce no ATP. Upon initiation of germination, the antibiotic is released from spores, leaving ATPase free from inhibition. It has been suggested that streptomycin is a component of the cell walls of producing S . griseus. This idea was tested using a mutant strain that produces streptomycin when streptidine is added to the medium. The rate and amount of growth was essentially the same when streptomycin was or was not produced. Cell walls from organisms grown in the presence or absence of streptidine were equally susceptible to lysozyme. Spores formed in both conditions were equally dormant and germinable. Cell walls from organisms growth with [14C]streptidinecontained label. This label was removed by a 1.0 M NaCl and was attributable to [14C]streptidine.Therefore, the antibiotic was ionically bound and not a structural component of the walls. No function for streptomycin at the level of cell walls was found (Ensing, 1980). The ticklish question of the role of secondary metabolites cannot be easily answered. The diversity of their chemical structure and biological activity suggests that the search for a general answer may be naive. Philosophical solutions to this problem were attempted many years ago. Although Waksman (1943, 1956) suggested that antibiotics are a metabolic waste product, these substances confer a competitive advantage upon the producing organism (Krassilnikov, 1958). B . brevis produces two types of peptide antibiotics: the linear gramicidins and cyclic tyrocidines. Most antibiotics that are produced during the early stages of sporulation are potent inhibitors of the vegetative growth of the producing organism. Gramicidin-negative mutants of B . breuis produce defective spores unless supplied with gramicidin within a critical period during transition from growth to sporulation. This period coincides with alteration in the specificity of transcription and suggests that peptide antibiotics may inhibit promoter recognition by RNA polymerase of genes required for veg-

100

VEDPAL SINGH MALIK

etative growth but not for sporulation (Sarkar et aZ., 1977). However, mutants that are defective in gramicidin S synthesis have been found to sporulate normally (Deniain and Piret, 1979). Sporulation is inversely related to increase in gramicidin titer. In a chemostat under carbon limitation, spores are formed at a dilution rate lower than that required for formation of gramicidin S synthetase. Antibiotic negative mutants sporulate well and most antibiotic-producing industrial strains are poor sporulators. Antibiotic synthesis and sporulation are two independent processes that compete for a common pool and respond to a common regulatory mechanism. The various aspects of secondary metabolism are very close to those of sporulation and production of oligopeptides antibiotics. Production of typical secondary metabolites is one of the earliest events in sporulation. There is a discrete point in time during outgrowth of spores at which spores overcome the inhibitory effects of the peptide antibiotics. In a medium lacking nitrogen, R . brevis cells at this stage undergo sporulation in the presence of both tyrocidine and gramicidin. In vitro tyrocidine binds to DNA and inhibits initiation of transcription. Gramicidin binds to RNA polymerase and inhibits transcription in the absence of tyrocidine. However, when both gramicidin and tyrocidine are present, gramicidin reactivates a tyrocidine-inhibited transcription system. In this way, both peptide antibiotics may play an antagonistic gene regulatory role during sporogenesis in B . breuis. Serine protease of Bacillus lichenijmmis releases antibiotic bacitracin in oitro. The extracellular proteases have been separated into three distinct serine enzymes on carboxymethyl cellulose (CMC) columns. Bacitracin forms a complex with the specific protease, CMC fraction I11 in uivo and inhibits the activity of the enzyme responsible for its activation in vitro. Coincidence chromatography and gel filtration experiments suggest a direct peptide-protein interaction. Bacitracin appears to exert product inhibitiontype control on the protease (Vitkovic and Sadoff, 1977a). An antibiotic was produced in vitro by proteolysis of the vegetative cell protein with the CMC 111protease. This antibiotic was shown to be identical to bacitracin in its UV spectra, biological activity, and chromatographic properties in a multiplicity of systems. Similarities exist between ethanol-extractable peptides excreted by sporulating cells and those produced in uitro, in their number, in their R f values on chromatography, and in their labeling pattern with D,L-[ 14C]ornithine. Crude bacitracin synthetase does not appear to contain nor docs it require CMC I11 protease for activity. Unequivocal involvement of peptide antibiotics in inducing sporulation in Bacillus would require genetic proof, because the catalytic concentration of the antibiotic sufficient to induce sporulation may be unmeasurable. Evidence accumulating in recent years casts doubt on the essential role of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

101

antibiotics for sporogenesis. Mutants of many organisms exist that sporulate well but produce no antibiotics (Demain, 1980). Antibiotics, like extracellular protease, are regarded as the markers of the first stage of sporulation, and in accordance with the theory of sequential induction are taken to be inducers of the next stage of sporulation. In no case, however, are sporulation and antibiotic synthesis quantitatively correlated. Many industrial strains of Streptomyces and antibiotic-producing molds sporulate poorly. Furthermore, selection of suitable culture conditions can intensify one of these processes at the expense of the other (Bernlohr and Novelli, 1960).

X. Epilogue Synthesis of a secondary metabolite is a highly ordered and coordinated event. Negative and positive regulatory control mechanisms operative during balanced growth have to be lifted, with the simultaneous accumulation and availability of initiating precursors. Concentration of acetyl, amino, amidino, and other group donors further influences the emerging metabolites. Mechanisms of secretion, and resistances to certain metabolites also have to be developed. Exploration of the molecular biology of secondary metabolism is now possible as a result of the availability of methods of recombinant DNA technology and reciprocal genetics. The changes in the transcription of genome responsible for initiation of secondary metabolism should be characterized. In the majority of cases, the enzymes catalyzing different stages of secondary metabolism are not known. Multiple enzymatic complexes are often involved in secondary metabolism and are not very stable. Work along these lines is in progress with representatives of the genera Bacillus, Streptomyces (Hopwood, 1980a; Malik, 1979b), yeast (Davis, 1980), and PenicilZium (Demain, 1980). Mutants that are deficient in proteases (Ohman et al., 1980) and nucleases may be used for isolating enzymes and plasmids from these organisms. Reciprocal genetics and recombinant DNA technology can circumvent the efforts required to understand genetics and molecular biology of industrial microorganisms and can yield most meaningful results that will contribute to the understanding of the regulation of the economically important metabolic pathways involved in secondary metabolism. HEFERESCES Aberhart, D. J. (1977). Tetrahedron 33, 1545. Abraham, E. P . , and Fawcett, P. (1974). In “Industrial Aspects of Biochemistry.” (B. Spencer, ed.), pp. 319334. North-Holland Publ., Amsterdam.

102

VEDPAL SINGH MALIK

Aharonowitz, Y. (1979). In “Genetics of Industrial Microorganisms.” (0.K. Sebek and A. I. Laskin, eds.). Amer. Soc. for Microbiology, Washington, D.C. Aharonowitz, Y. (1980). Annu. Rev. Microbial. 34, 209. Aharonowitz, Y . , and Demain, A. L. (1978). Antimicroh. Agetits Chemothw 14, 159. Aharonowitz, Y., and Demain, A. L. (1979). Can. J . Microhiol. 25, 61. Aharonowitz, Y., and Demain, A. L. (1980). Biotechnol. Bioeng. 22 (Suppl. l), 5. Akagawa, H., Okaniski, M., and Umezawa, €1. (1975). J. Gen. Microbiol. 90,336. AliKhanian, S. I., and Danilenko, V. N. (1979). Hind. Antihiof. Bull. 21, 125. AI’Nuri, M. A , , and Egorov, N. S. (1968). Mikrobiologiya 37, 413. Alsberg, C. L., and Black, 0. F. (1912). Proc. Soc. E x p . Biol. M e d . 9, 6. Alton, N. K., arid Vapnek, D. (1979). Nature (London) 282, 864. An, G., and Vining, L. D. (1978). Can. J. Microbiol. 24, 502. Aoki, H., and Oktthara, M. (1980). Annu. Reu. Microbiol. 34, 159. Argoudelius, A. D., and Mason, D. J . (1969). J . Antibiot. 22, 289. Arima, K., Okazaki, H., Ono, H., Yarnada, K., and Beppu, T. (1973). Agric. B i d . Chem. 37, 2313. Arnstein, H. R. V., and Morris, L). (1960). Biochem. J. 76, 357. Arnstein, H. R. V.. Artman, N., Morris, D., and Toms, E. J. (1960). Biochem. J. 76, 353. Atkinson, D. E., and Walton, G. M. (1967). J . Biol. Chern. 242, 3239. Audhya, T. K., and Russell, D. W. (1975). J . Gen. Miwohiol. 86, 327-332. Baldwin, J. E., Singh. P. D., Yoshida, M., Sawada, Y., and Demain, A. L. (1980). Biochem. J. 186, 889. Ball, W. J . , and Atkinson, D. E. (1975). J. Bacteriol. 121, 085. Bandyopadhyay, S . K., and Majumdar, S. K. (1974). Antirniwob. Agents Chernuther. 5,431. Banerjee, A. B., and Bose, S. K. (1974). J . Ractcriol. 87, 1397-1406. Barahas, G. (1980). Folia Microbiol. 25, 270. Basak, K., and Majumdar, S. K. (1973). Anfirnicrob. Agents Chernother. 4, 6. Battersby, A. H. (1978). Ezperientia 34, 1-13. Behal, V., and Vanek, Z. (1970). Folia Micrubiol. 15, 354. Behal, V., Cudlin, and Vanek, Z. (1969))). Folia Microbiol. 14, 117. Behal, V., Jechova, V., and Vanek, Z. (1977). Phytochernistry 16,347. Benedict, R. C., Lindenfelser. L. A , , Stodola, F. H., arid Traufler, D. H. (1951). J. Bacteriol. 62, 487. Bentley, R., and Campbell, I. M. (1968). I n “Comprehensive Biochemistry; Metabolism of Cyclic Compounds” (M. Florkin and E. H. Stolz, eds.), Vol. 20, p. 415. Elsevier, Amsterdam. Bentley, R., and Keil, J. G. (1962). J . B i d . Chern. 237, 867. Bcnveniste, R., and Davies, J. (1973). Proc. Natl. Acad. Sci. U . S . A . 70, 2276. Berdy, J . (1974). Adu. Appl. Microbiol. 18, 309406. Berdy, J. (1980). Process. Biochem. 15(7), 28. Bernlohr, R. W., and Novelli, G. D. (1960). Arch. Biochern. BiophyY. 87, 232. Bernlohr, R. W., and Novelli, 6. D. (1963). Arch. Biochem. Biophys. 103, 94-104. Bibb, M. J., Ward, 1. M., and Hopwood, D. A. (1978). Nature (London) 274, 398. Bibh, M. I , , Schottel, J . , and Cohen, S. N . (l98Oa). Nature (London) 284, 526. Bibb, M. J.. Ward, J. M.,and Hopwood, D. A. (1980b). Dcu. Inn. Microbiol., in press. F.I IW. , (1953). Aust. J. Cheiri. 6, 360. Birch, A. J., and D O ~ O V ~ Birch, A. J., Massey-Westropp, R. A,, and Moye, C. J. (1955). Ailst. J . Chem. 8, 539. Birkinshaw, J. H., Oxford, A. E., a d Raistrick, H . (1936). Biochem. J. 30, 394. Block, K., and Vance, D. (1977). Antzu. Reo. Biochem. 46, 2m-293. Blumauerova, M., Mateju, J., Stajner, K., and Vanek, Z. (1977). Folia Mimobid. 22, 275.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

103

Blumauerova, M., Podojil, M., Gauze, G. F., Maksimova, T. S., Panos, J., and Vanek, Z. (1980a). Folia Microbid. 25, 207. Blumauerova, M . , Podojil, M., Gauze, G. F., Maksimova, T. S., Panos, J., and Vanek, Z. (1980h). Folia Mimohiol. 25, 213. Bollon, A. P . , and Vogel, H. J. (1973). J. Bacteriol. 114, &32. Borisoglebskaya, A. N . , Perebityak, A. N., and Boronin, A. M . (1979). Antibiotiki 24, 883. Boronin, A. M . , and Sadovnikova, L. G . (1972). Genetics (USSR) 8, 174. Borowska, Z. K., and Szer, W. (1976). Biochiin. Biophys. Acta 418, 63. Borrow, A., Jeffreys, E. G . , Kessell, R. H. J., Lloyd, E . C., Lloyd, P. B., and Nixon,, I. S . (1961). Can. J. Micohiol. 77, 222. Borrow, A , , Brown, S . , Jeffreys, E. G . , Kessell, R. H. J . , Lloyd, E . C., Lloyd, P. B., Rothwell, A., Rothwell, B., arid Swait, J. C. (1964). Can. J. Mimohiol. 10, 407. Borrow, A., Brown, S., Jefferys, E. G . , Kessell, R. H. J., Lloyd, E. C., Lloyd, P. B., Rothwell, A., Rothwell, B., and Swait, J . C. (l964b). Can. J. Mimohiol. 10, 445. Bose, R . , Ray, B., and Bose, S . K. (1979). Foliu Mirruhiol. 24, 373. Brack, A. (1947). IZela. Chiin. Actu 30, 1. Brandsch, R. Hefco, E., Brandsch, C . , Rotinberg, P., and Kelemen, S. (1980). J. Antibiot. 33, 1204. Brockinann, H., Zeeck, A,, Menve, A., and Muller, W. (1966). Justus Liehigs Ann. Deo. Chem. 698, 209-229. Bruehl, G . W . , Millar, R. L., and Currfer, B. (1969). Can. J. Plant Sci. 49, 235. Bu’Lock, J . C. (1961). Ado. A p p l . Microhiol. 3, 293. Bu’Lock, J. D. (1967).“Essays in Biosynthesis and Microbiol Development. Wiley, New York. Bu’Lock, J . D. (1974). “Industrial Aspects of Biochemistry,” (A. J. Spencer, ed.), Vol. 1. NorthHolland Puhl., Amsterdam. Bu’Lock, J . D. (1975a). Dec. l n d . Microhiof. 16, 11. Bu’Lock, J. D. (1975h). 111 “The Filameritous Fungi” (J. E. Smith and D . R. Berry, eds.), Vol. 1, pp. 33-58, Wiley, New York. Bu’Lock, J. D., Hainilton, D., M U ~ I WM. , A,, Powell, A. J . , Slnalley, H . M.,Shepherd, D., and Smith, G . N. (1975). Can. J . Microhiol. 11, 765-778. Byiig, 6. S . , and Turner, J. M. (1977). Biochetn. J . 164, 139. Cameron, J. R., Loh, E. Y . , and Davis, R. W. (1979). Cell 16, 739. Cape, R. E. (1979). CHEMTECZZ 638. Carson, M . , and Jensen, R. A . (1974). J . Bacteriol. 117, 312. Catlin, E. R., Hassall, C. H . , and Pratt, B. C. (1968). Biochim. Bioph!/s. Acta 156, 109. Cella, R., and Vining, L. C. (1975). Cun. J. .Vfic.rohiol. 21, 463. Chain, E. B. (1980). CHEMTECII. Chalcff, D. T., and Fink, G . R. (1980). Cell 21, 227. Chandler, M.,Boydelatorir, E . , Willeins, D . , and C h o , L,. (1979). A40l. Gen. Genet. 176, 221. Chandra, P . , Read, G . , and Vining, L. C. (1966). Can. J. Biochein. 44, 403. Chang, L. T., Behr, D. A., and Elander, R. P. (1980). Deo. Inn. hficrobiol. 21, 233. Chapman, A. G., Fall, L., and Atkinson, D. E. (1971). J . Bacteriol. 108, 1072-1086. Chassy, B. M., and Suhadolnik, R. J . (1969). Biochiiti. Bioph!ys. Actu 182, 307. Chatrr, K. F. (1980). Dco. lnrl. Microbial. 21, 65-74. Chater, K. F., and Merrick, M . J. (1979). I n “llevelopmental Biology of Prokaryotes” (J. H . Parish, ed.). Blackwell, Oxford. Chopra, I., and Howe, T. G. 9. (1978). Microhiol. Reo. 42, 707-724. Cochrane, V. W. (1958). “Physiology of Fungi,” p. 308. Wilry, New York. Cocito, C. (1979). Microhiol. Ruo. 43, 145. Cohen, S . , and Shapiro, J. A. (1980). Sci. A ~ R 242, . 40.

104

VEDPAL SINGH MALIK

Collie, J. N. (1893). J . Chetn. Soc. 329. Collie, J. N. (1907). J. Chem. SOC. 1806. Chrcoran, J . W. (1974). I n “Genetics ofIndustrial Microorganisins” (2. Vanek, Z. Hostalek, and J. Cudlin, etls.) Vol. 2, pp. 339351. Academia, Prague. Crawford. I. P., and Stauffer, C . V. (1980). Annu. Rev. Biochem. 49, 163. CundlLT, E . (1978). Suture ( . h 7 d 0 7 1 ) 272, 7%. Chidlitf, E., and Tlio~npson,J. (1979). Nature ( L o d o n ) 278, 859, II’Amato, R. F., and Pisano, M . A. (1976). Antonic oun Lrezweithoek J . A4ic~obiol.Serol. 42, 299308, Davis, R . D. (1980). Science 209, 78. Davies, J , , and Srnith, D. I. (1978). Anriu Reo. ~Microl>bl.32, 468. Davics, J . , Houk, C., Yagisawa, M., and White, T. J . (1979). I n “Genetics of Industrial Microorganisms” (0. K . Sehek and A. I. Laskin, eds.). Airier. Soc. Microbial., Washington, D.C. Deniain, A. L. (1963). Clin. Med. 70, 2045-2051. Dnmain, A. L. (1966). Ado. Appl. Micwbiol. 8, 1. Deinaiii, A. L. (1968). L~oydirr31, 395418. Deinain, A . L. (1972). J . A p p l . C h e m Biotechttol. 22, 345. Demain, A. L. (1973). Ado. A p p l . Microbiol. 16, 177. Deinain, A. I,. (1974a). Ann. N . Y . Acnd. Sci. 235, 601. Demain, A. L. (1974b). Llt~ydin37, 147. Deinain, A. L. (1980). Seurch 11, 148. Deinain, A. L., antl Rirnhaum, J. (1968). Crcrr. Top. Microbiol. l m n n n o l . 46, 1. Demain, A. I , . , and Inamine, E. (1970). Bacteriol. Reo. 34, I . Demairi, A. L., and Kennel, Y. M. (1978). J. Ferment. l’echnol. 56, 323. IIt.inain, A. L., and Masurekar, P. (1974). J . Gen. Mitxohiol. 82, 143. Deniain, A . L., and Piret, J. (1979). In M . Luckner and K. Schrieber, eds.), pp. 183-188. “Regulation of Secondary Product and Plant Hormone Metabolism. Pergamon, Oxford. Demain, A. L., Newkirk, 1. F., and IIendlin, D. (1963). J. Bucteriol. 85, 339. DeMoss, H. D. (1967). In “Antibiotics. Violaccin” (D. Gottlieb and P. D. Shaw, eds.), Vol. 2, pp. 77-81. Springer-Verlag, Berlin arid New York. Ikshpaiidr, V. N . (1968). H i n d Antibiot. Bull. 11, 106-112. Dcuel, T. F., Ginsberg, A., Yeh, J., and Shelton, E., and Stadtman, E. R. (1970). J . B i d . Chern. 245, 5195. Diaz, J. C., and Clowes, R. C. (1980). J. Bacteriol. 141, 1015. Dimroth, P., Ringelmann, E., and Lynen, F. (1976). hhr. J . Biochern. 68, 591. Dowding, J . E. (19794. J. Gen. Micwbiol. 115, 385. Dnwding, J. E. (197911). F E M S M i c w b . Lett. 6, 9 5 Doy, C. I I . (1968). Reo. Pure A p p l . Chem. 18, 41. Drew, S. W., and Demain, A. L. (1975a). Antirnicroh. Agents Chemother. 8,s-10. Drew, S. W., and Dernain, A . L. (197%). Eur. J . Appl. A!icrol?iol. 2, 121. Drew, S. W.. and Demain, A. L. (1975~).J. Antibiut. 28, 889-895. Drew, S. W., antl Demain, A. L. (1977). Annu. Rev. it4icrobiol. 31, 342356. Dubois, E. M . , Grenson, A., and Wiatne, J. M . (1974). Eur. J . Biockein. 48, 603-616. Dulaney, E. 1,. (1948). J . Bucteriol. 56, 305313. Dulancy, E. L., and Dulaiiey, D. D. (1967). Truns. N . Y. A m d . Sci. 29, 782. Eble, T. (1978). J . Chroinutogr. L i b . 15, 231-271. Egorov, N . S . , Toropova, E. G . , and Suchkova, L. A. (1971). MikrolJiologiyu 40, 475. Ehrensvard, G . (1955). E x p . Cell Res. Suppl. 3, 102. Elander, K. P.. Mabe, J. A.. and Gorman, M. (1970). A p p l . Microhiol. 19, 721.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

105

Elander, R. P., Mabe, J. A., Hamill, R . L., and Gorman, M. (1971). Folia Microbiol. 16, 156-165. Elson, E. W., and Oliver, R. S . (1978). 1. Antibiot. 31, 586. Elstner, E. F., and Suhadolnik, R. J. (1971). J. Biol. Chem. 246, 6973. Elstner, E. F., and Suhadolnik, R. J. (1975). Methods Enzymol. 43, 515-520. Englesherg, E., and Wilcox, G. (1976). Annu. Rev. Genet. 8, 219-242. Ensing, J. C. (1978). Annu. Reu. Microbiol. 32, 185. Fadeeva, L. E., and Malkov, M. A. (1978). Aritibiotiki 23, 1061. Farley, J. R., Mayer, S., Chardler, C. J., and Segel, I. H . (1979). J. Bacteriol. 137, 350. Fisher, P. B. (1980). Process. Biochem. 15, 2. Flickinger, M .C., and Perlman, D. (1979). Antimicrob. Agents Chemother. 15, 282. Floss, H. G., Robbers, 1. F., and Heinstein, P. F. (1974). Rec. Adu. Phytochem. 8, 141. Fontana, R . , Canepari, P., Satta, C . , and Coyette, J. (1980). Nature (London) 287, 70-72. Foster, J. W. (1949).“Chemical Activities of Fungi,” pp. 164-169. Academic Press, New York. Foster, J. W., and Waksman, S . A. (1939). J . Bacteriol. 37, 599. Francis, M.,Cella, R., and Vining, L. C . (1975). Can. 1. Microbiol. 21, 1151. Francis, M. M., Vining, L. C., and Westlake, D. W. S . (197th). J. Bacteriol. 134, 10-16. Francis, M. M., Vining, L. C., and Westlake, D. W. S. (l978b). J. Bacteriol. 134, 10. Freeman, R. F . , and Hopwood, D. A. (1978). J. Gen. Microhiol. 106, 377. Freeman, R. F., Bibb, M. J., and Hopwood, D. A. (1977). 1. Gen. Microbiol. 98, 453. Freese, E., Heinze, J. E., and Galliers, E. M. (1979). J. Gen. Microbiol. 115, 193. Friebel, T. E., and Demain, A. L. (1977a). J. Bacteriol. 130, 1010-1016. Friebel, T. E., and Demain, A. L. (1977b). FEMS Mi~robiol.Lett. 1, 215-218. Friedrich, C. G., and Demain, A. L. (1977). 1.Antibiot. 30, 760-761. Friend, E. J., Warren, M . , and Hopwood, D. A. (1978). J. Gen. Microbiol. 106, 201-206. Froyshov, 0. (1974). FEBS Lett. 44, 75. Froyshov, 0. (1977). FEBS Lett. 81, 315318. Froyshov, O., and Laland, S. G. (1974). Eur. 1.Biochem. 46, 235. Fujikawa, K., Suzuki, T., and Kurahashi, K. (1968). Biochetn Biophys. Acta 161, 232. Fujiwara, T., Tanimoto, T., Matsumoto, K . , and Kondo, E. (1978). 1.Antibiot. 31, 966-969. Funk, A , , and Divekar, P. V. (1959). Can. J . MicrohioZ. 5, 317321. Fynn, G. H., and Davison, A. C . (1976). 1. Gen. Microbiol. 94, 68-74. Gallo, B. J. (1979). Int. Symp. Genet. Ind. Microorg. 3rd, Madison, Wisconsin 1978 Abstr. No. 35. Gallo, M., and Katz, E. (1972). J. Bacteriol. 109, 659467. Garcia, E., Rancruft, S. Rhee, S. G., and Kustu, S. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 1662. Gatenbeck, S., and Hermodsson, S. (1965). Acta Chem. Scand. 19, 65. Gatenbeck, S., and Sjoland, S. (1964). Biochim. Biophys. Acta 93, 246. Gaworowska, M. J., Emilianowicz, C. S., Switalski, L., and Raczynska, B. K. (1975).Antimicrob. Agents Chemother. 8, 526. Gersch, D. (1980). Process. Biochem 15, 21. Gerscli, D., Romer, W., Bocker, H., and Thrum, J . (1978). FEMS Microbiol. Lett. 3, 39. Gersch, D., Skurk, A . , and Romer, W. (1979). Arch. Microbiol. 121, 91. Ghisalha, O., and Nuesch, J. (1978a). J. Antibiot. 31, 215. Ghisalba, O., and Nuesch, J. (1978b). 1. Antibiot. 31, 202. Gibson, F., and Pittard, J. (1968). Bacterial. Reu. 32, 465. Gifiorn, F . , and Goltschalk, G. (1975). J. Bacteriol. 124, 1052. Ginther, C. L. (1979). Antimicrob. Agents Cheniother. 15, 522. Godfrey, J. C., and Price, K. E. (1972). Adu. Appl. Microbiol. 15, 231.

106

VEDPAL SINGH MALIK

Godfrey, 0.(1973). Antitriimoh. Agents Cheinothw. 4, 73. Goldberg, N . D., and IIaddox, M. K. (1977). Annu. Reo. Biocheiti. 46, 823396. Goldhcrger, R. F., etl. (1979). “Biological Regulation and Development,” Vol. 1. Plenum, New York. Gordee, E., and Day, L. E. (1972). Antiinicwh. Agents Chemother. 1, 315. Gordee, E. Z., and Day, L. E. (1972). Antiinicrob. Agents Cheinother. 1, 315. Gorisch, 11.. and Ligens, F. (1972). Arch. h4ikrobiol. 82, 147. Gorisch, H . , a i d Lingelis, F. (1974). Biochemistry 13, 3790. Gorman, M., and Huber, F. M. (1979). Annu. Rep. Ferment. Proc. 2, 203. Gottlieh, S. (1976). J. Antikiot. 29, 987. Goulden, S. A., and Chattaway, F. W. (1968). Biocheiii. J. 110, 550. Gouldcn, S. A . , and Chattaway, F. W. (1969). /. Cen. Mic7ubio1. 59, 111. Gourevitch, A., Pursiano, T. A., and Lein, 1. A. (1961). Arch. Biochetn. Biophys. 93, 283. Grafe, U., Bocker, H., Reinhardt, G., Tkocz, I I . , and Thrum, H. (1974). Z. M g . Mikrohiol. 14, 181. Grafe, U . , Bocker, H . , and Thrum, H. (1977). 2. Allg. Mikrobiol. 17, 201. Grafe, Y.. Bocker, H . , Reinhardt, G . , and Thrum, H. (1974). Z. Allg. Mikrobiol. 14, 659. Graham, M . Y., and Weisbluirr, B. (1979). J . Bucteriol. 137, 1464. Greer, H . , and Fink, C . R. (1979). Proc. N a t l . Acad. Sci. U . S . A . 76, 4006. Gregory, K. F., and Huang, J . C . C. (1964). J. Bacterial. 37, 1287. Grnotwasaink, 1. W., and Gaucher, G. M. (1980). J. Bacteriol. 141, 443. Haavik, H . I. (1974). J . Cen. Microbiol. 84, 321-326. Haavik, H.1. (1979). Folia Mitrobiol. 24, 365. Hachimori, A . , h4atsunanga, A , , Shirnizu, M., Samejirna, T., and Nosoh, Y. (1974). Biochim. Biophys. Acta 350, 461. Hayakawa, T., Tanaka, T., Sakaguchi, K., Otake, N., and Yonehara, H . (1979). J . Gen. Appl. Microhiol. 25, 255. Helser, T. E., Davies, J , E . , and Dahlhcrg, J. E. (1971). Nuture (London)New B i d . 233, 12. Herrdlin, D. (1949). Arch. Biochem. Bioph!ls. 24, 435. IIinnen, A,, and Nuesch, J. (1975). Antimicrub. Agents Chemother. 9, 824. Hitchcock, M. J. M . , and Katz, E. (1978). Antimimob. Agents Chernother. 13, 104-114. Hodgson, B. (1970). J . Thew. B i d . 30, 111-119. Hodgson, D. A . (1980). Ph.D. Thesis, University of East Anglia, England. Iioeksema, H., and Krueger, W. C. (1976). J. Antibiot. 29, 704-709. Hoeksema, H., and Smith, C. G. (1961). Prog. I t i d . Microhiol. 3, 91-139. Hoeksema, H . , Mizsak, S . , and Baczynsky, J. (1979). J. Aiztihiot. 22, 773-776. Holtje, J. V. (1978). Eur. J , Biochein. 86, 345. Holtje, J. V. (1979). Antimimob. Agents Cheniotht-r. 15, 177. Holzer, H . , and IIeinrich, P. C . (1980). Annu. Rev. Biochem. 49, 63. Hook, D. J , , Chang, L. T., Elander, R. P., and Morin, R. B. (1979). Biochem. Biophys. Res. Conmun. 87, 258. Hopwood, U. A. (1978). Annu. Rel;. Microbiol. 32, 373392. Hopwood, D. A. (1980). Int. S y n p . Aclinomycete Biol. Hopwood, D. A. (1980b). I n “Antibiotics of the Future” (L. Ninet, ed.). Academic Press, New York. IIopwood, D. A., and Chater, K. F. (1980). Philos. Trunu. R . Sac. London Ser. B 290, 313. Hopwood, D. A , , and Merrick, M. J. (1977). Bacteriol. Reo. 41, 595-635. Horinouchi, S., and Weishlum, B. (1980). Proc. Natl. Acad. Sci. U . S . A . 92. Horner, W. H . (1964). Proc. Natl. Acad. Sci. U . S . A . 67, 305.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

107

Hostalek, Z. (1964a). Folia Microbiol. 9, 78. Hostalek, 2. (1964b). Folio Microhiol. 9, 89. Hostalek, Z. (1Wc). Folia M i ~ r o b i o l .9, %. Hostalek, Z. (1979). Folia Mimobiol. 25, 445. Hostalek, Z., Tinterova, M . , Jechova, V., Blumauerova, M., Suchy, S., and Vanek, Z. (1969). Biotechnol. Bioeng. 11, 539. Hostalek, Z . , Tobek, I., Bobyk, M. A., and Kulayev, I. S. (1976). Folia Microhiol. 21, 131. Hostalek, Z., Blumauerova, M . , and Vanek, Z. (1979). Econ. Microbiol. 3, 293. Hotta, K., Okami, Y., and Umezawa, H. (1977). J . Antibiot. 30, 1146. Howells, J. D., Anderson, L. E., Coffey, G. L., Senos, 6. D., Underhil, M. A., Vogler, D. L., and Erlich, J. (19’72). Antirnicrob. Agents Chemother. 2, 79. Huang, M . , and Gibson, F. (1970). J. Bacteriol. 102, 767. Hughes, P. F., Troost, T., and Katz, E. (1977). Abstr. Annu. Meet. Am. Sac. Mi~robiol.p. 253. Hurley, L. J., and Bialek, D. (1974). J . Antihiot. 27, 49. Hurst, A. (1966). J . Cen. Microbiol. 45, 503. Hutchison, K. W., and Hanson, R. S. (1974). J. Bacteriol. 119, 70-75. Hutter, R . , Kieser, T., Crameri, R . , and Hintermann, G. (1979). Proc. Int. S!ynp. Actinoniycete B i d . Cologne. Imsande, J. (1978). Microbiol. Rev. 42, 67-83. Inamine, E., Lago, B. D., and Demain, A. L. (1969). In “Fermentation Advances” (D. Perlman, ed.), pp. 199-221. Academic Press Inc., New York. Ishihara, H . , Endo, Y . , Abe, S., and Shimura, K. (1975). FEBS Lett. 50, 4346. Ito, M . , Aida, J . , and Uemura, T. (1969). Agric. B i d . Chem. 33, 262-269. Ito, M., Aida, K., and Vermura, T. (1970). Proc. Int. Congr. Chemother. 6th p. 1128. Janglova, Z., Suchy, J . , and Vanek, Z. (1969). Folia Microbiot. 14, 208. Jarvis, F. G., and Johnson, M. J. (1947). J . Am. Chern. Soc. 69, 3010. Jensen, R. A,, and Nasser, D. S. (1968). J. Bacteriol. 95, 188. Johnson, M. J. (1952). Bull. WHO 6, 99-121. Johnston, H. M., Barnes, W. M . , Chumley, F. G., Bossi, L., andRoth, J . R. (1980). Proc. h’atl. Acad. Sci. U . S . A . 77, 508. Jones, A , , and Vining, L. D. (1976). Can. J . Mimohiol. 22, 237-244. Jones, A., and Westlake, D. W. S. (1974). Can. J . Microbial. 20, 1599. Jones, A., Francis, M., Vining, L. C., and Westlake, D. W. S. (1978). Can. J . Microbiol. 24, 238. Jones, G. H . (1975). Biochem. Biophys. Res. Commun. 63, 469. Jones, G. H. (1979). Arch. Biochem. Biophys. 198, 195. Jones, G. H., and Weissbach, J. (1970). Arch. Biochem. Biophys. 137, 558-573. Kahan, J. S., Kahan, F. M . , Goegelman, R., Currie, S. A., Jackson, M . , Stapley, E. O., Miller, T. W. Hendlin, D., Mochales, S., Hernandez, S., Woodruff, H. B . , and Birnbaum, J . (1979). J . Antibiot. 32, 1. Kalakoutskii, L. V., and Agre, N . S . (1976). Bacteriol. Rev. 40, 469. Kane, J., and Jensen, R. A. (1970). J . Biol. Chem. 245, 2384. Kane, J. F. (1975). Arch. Biochem. Biophys. 170, 452. Kane, J. F., Holmes, H. W., and Jenson, R. H. (1972). J . B i d . Chern. 247, 1587. Katz, E. (1967). In “Antibiotics” (D. Gottlieb and P. D. Shaw, eds.), Springer-Verlag, Vol. 2. Berlin and New York. Katz, E. (1968). Lloydia 31, 364. Katz, E., and Demain, A. L. (1977). Bacteriol. REV. 41, 449474. Katz, E., and Weissbach, H. (1963). J . Biol. Chem. 238, 666-675.

108

VEDPAL SINGH MALIK

Kauffman, G. B. (1979). J. Chenz. Ecluc. 56, 454. Kawaguchi, A,, Tomodo, H., Okudo, S . , Awaya, J . , and Omura, S . (1979). Arch. Biochem. Biopluls. 197, 30. Keilman, G. R . , Burtis, K., Tanimoto, B., and Doi, R. 11. (1976). J. Bacteriol. 128, 8 0 4 5 . Keller, E. B., and Calvo, J . M. (1979). Proc. Natl. Acud. Sci. U . S . A . 76, 6186. Kennel, Y. M., and Deinain, A. L. (1978). E x p . Mycd. 2, 234-238. Khokhlov, A. S., and Tovarova, I. I. (1972). Postepy Hig. Med. Dosw. 26, 469491. Khokhlov, A. S., and Tovarova, I. I. (1979). In “Regulation of Secondary Product and Plant Hormone Metabolism” (M. Luckner and K. Schreiber, eds.), pp. 133-145. Pergainon, Oxford. Khokhlov, A. S . , Anisova, L. N., Tovarova, I. I . , Kleiner, E. M., Kovalenka, I. V., Krasilnikova, 0. I., Kornitskaja, E. Y., and Pliner, S.A. (1973). Z. Allg. Mikrobiol. 13,647455. Kibby, J . , McDonald, L. A , , and Richards, R. (1980). J . Chem. Soc. Cherra. Cornin. 768. Kida, M., Asako, T., Yoneda, M . , and Mitsuhashi, S. (1975). In “Microhiol Drug Resistance” (S. Mitsuhashi and H. Hasimoto, eds.). Univ. Park Prcss, Baltimore, Maryland. Kieser, T., Hinterinan, G., Crameri, R., and Hutter, R. (1979). Proc. Znt. Symp. Actinoitiycefe HiO/.

C
Kimura, A. (1967). Agric. B i d . Chrm. 31, 845-852. Kirby, J. P. (19880). Processs.Bioclicm. 15(7), 14. l . 3, 283. Kirby, R . (1978). F E M S M i ~ ~ o h i oLett. Kirsch, E. J. (1967). I n “Antibiotics” (D. Gottlieh and P. U . Shaw, eds.). Vol. 2. SpringerVerlag, Berlin and New York. Klein, H. L., and Welch, S. K. (1980). Nucleic Acids Hes. 8, 4651. Klciner, E. M., Pliner, S. A., Soifer, V. S . , Onoprienko, V. V., Balashova, T. A . , Hozynov, B. V., and Khokhlov, A. S. (1976). Bioorg. Khirn. 2, 1142-1147. Kleinkauf, H . , and Gcvers, W. (1969). Cold Spring Harbw S!/irap. Quant. B i d . 34, 805-813. Kleinkauf: H . , and Koischwitz, H. (1974). I n “Liptnann Symposiun: Energy, Biosynthesis and Regulation in Molecular Biology,” pp. 336344. De Gruyter, New York. o l 600. . Klucpfel, D., Laiicini, G. C., and Sartori, G . (1965). Appl. M i ~ ~ ~ b i13, Kniep, B., and Grisebach, H. (1976). FEBS Lett. 65, 44. Kniep, B., and Grisehach, H. (1980). .I. Antihiot. 33, 416. Koch, G . , and Richter, D., eds. (1979). “Regulation of Macromolecular Synthesis by Low Molecular Wright Mediators.” Academic Press, New York. Kogt*kai-,R . C . , and Deshpande, V. N . (1979). Biochinz. Biophys. Acta, 133, 37. Kojima, M., Yainada, Y . , and Vrnezawa, 1%.(1969). Agric. B i d . Cherrt. 33, 1181. Komatsn, K., and Kodaira. R. (1977). J. Antihiot.‘30, 226. Kominek, L. A. (1972). Antiiiiicrob. Agents C h i o t h e r . 1, 123. Kominrk, L. A. (1975). Antimicrob. Agerrt.9 Cheinother. 7, 861. Kralovcova, E., Blumauerova, M., and Vanek, Z. (1977). Folio Microbial. 22, 182. Krassihiikov, N. A. (1958). Acutl. Sci. USSR (Moscow) p. 365. Krrhs, G., a d Beavo, J. A. (1979). Annu. net.. Biocheiu. 48, 923. Krupinsky, V. M . , Robbers, J. E . , and Floss, H. G. (1976). J . Bacteriol. 125, 158. Kuruhashi, K . , Yainada, M., Mori, K., Fujikawa, K., Kainbe, M . . Imae, E., Takahashi, II., and Sakamoto, Y. (1969). Cold Spring Harhor S!ynap. Quunt. B i d . 34, 815. Kurylo-Borowsky, Z. (1967). Z I L “Antibiotics” (D. Gottlieh and P. D. Shaw, eds.), Vol. 2, pp. 342352. Springer-Verlag, Berlin and New York. Kurylowicz, W., and Kowszyk-Gindifer, Z. (1979). Ifind. Antibiot. Bull. 21, 115. Lagosky, P. A., a i d Chany, F. N. (1980). J . Bacteriol. 144, 499. Laland, S. G., and Zirnincr, T. L. (1973). I.:,wuys Biochrin. 9, 31-57. Lancini, 6. C., and White, R. C. (1973). Process. Biochcm. 8, 14-16.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

109

Laskowski, M., and Kato, I. (1980). Annu. Reo. Biochem. 49, 593. Lawrence, J., Cox, G. B., and Gibson, F. (1974). J . Bacteriol. 118, 41. Lee, S. G., Littau, V., and Lipmann, F. (1975). /. Cell B i d . 66, 238. Lemke, P. A , , and Nash, C. H . (1972). Can. J . Microbiol. 18, 255. Liddel, J. M., Shaw, W. V . , and Swan, I. D. A. (1978). J . Mot. Biol. 124, 285. Lipmann, F. (1971). Science 173, 875-884. Liras, P., Villanueva, J. R., and Martin, J. F. (1977). J . Gen. Microhiol. 102, 269. Lisivinova, S. N., Dmitrieva, S. V., Zaslavskaya, P. L., and Orlova, N . V. (1979). Antibiotiki 24, 888. Liu, C . M., McDaniel, L. E., and SchlafFner, C. P. (1972). J . Antibiot. 25, 116. Liu, C. M., McDaniel, L. E., and SchlafFner, C. P. (1975). Antimicrob. Agents Chemother. 7, 196-202. Liu, C. M., Hermann, T., and Miller, P. A. (1977). J . Antihiot. 30, 244-251. Liu, C., Hermann, T. E., and Miller, P. A. (1979). J . Antibiot. 32, 1025. Lockwood, L. B., and Nelson, G. E. N . (1946). Arch. Biochem. 10, 365. Lomovskaya, N. D., Chater, K . F., and Mkrtumian, N. L., (1980). Microbiol. Rev., 44, 206. Longley, R. P., Halliwell, J. E., Campbell, J. J. R., and Ingledew, W. M. (1972). Can. J . Microbiol. 18, 1357. Losick, R. (1981). In “Molecular Cloning and Gene Regulation in Bacilli.” Academic Press, New York. In press. Lowe, D. A , , and Westlake, D. W. S. (1971). Can. J . Biochem. 49, 448. Lowe, D. A , , and Westlake, D. W. S. (1972). Can. J . Biochern. 50, 1064. Luckner, M . , Nover, L., and Bohm, U . (1977). “Secondary Metabolism and Cell Differentiation.” Springer-Verlag, Berlin and New York. Luengo, J. M.,Revilla, G., Villanueva, J . R., and Martin, J. F. (1979). J . Gen. Microbid. 115, 207. Luengo, J. M., Revilla, G., Lopez, M. J., Vdlanueva, J. R., and Martin, J . F. (1980). J. Bacteriol 144, 869. Lurie, L. M., Verkhotseva, T. P., and Levitov, M . M. (1975). Antibiotiki (Moscow)20, 291. Magasanik, B., Prival, M., Brenchley, J . , Tyler, B., Deleo, A , , Streicher, S., Bender, R., and Paris, C. (1974). Cum. Top. Cell Reg. 8, 118. Maggon, K. K . , Gupta, S. K., and Vankitasubramanian, T. A. (1977). Bacteriol. Reu. 41, 822-855. Magni, G . , Pallotta, G . , Natdhni, P., Ruggieri, S., Santarelh, I., and Vita, A. (1978). J . Biol. Chem. 253, 2501. McCray, J. W., and Hermann, K. M. (1976). J . Bacteriol. 125, 608. McGuire, J. C . , Glotfelty, G., and White, R. J. (1980). FEMS Mimobiol. Lett. 9, 141. Maier, W., Erge, D., and Groger, D. (1980). Planta Mecl. 40, 104. Majumdar, M. K., and Majumdar, S. K. (1970). Biochem. J . 120, 271-278. Majumdar, M . K., and Majumdar, S. K. (1971). Fotia Microbiol. 16, 285-292. Majumdar, M. K., and Majumdar, S. K. (1972). Biochem. J . 122, 397-404. Matins, J. F., Normansell, I. D., and Holt, G . (1979). 1.Gen. Microbiol. 112, 113. Malik, V. S. (1972). Arlzj. A p p l . Microbiol. 15, 297-336. Malik, V. S. (1977). J . Antibiot. 30, 897-899. Malik, V. S . (197%). Ado. Genet. 20, 37. Malik, V. S. (1979h). Ado. Appl. Microbid. 25, 75. Malik, V. S. (1980a). Trends Biochem. Sci. March, p. 82. Malik, V. S. (1980b).Ado. Appl. Microbiol. 27, 1. Malik, V. S . (1980~).Biotechnol. Lett. 2, 455. Malik, V. S., and Reusser, F. (1979). Plusinid 2, 627.

110

VEDPAL SINGH MALIK

Malik, V. S., and Vining, L. C. (1970). Can. J . Microbiol. 16, 173. Malik, V. S . , and Vining, L. C. (1971). Can. J . Microbiol. 17, 1287-1290. Malik, V. S., and Vining, L. C. (1972a). Can. J. Microbiol. 18, 137. Malik, V. S., and Vining, L. C. (1972b). Can. J. Microhiol. 18, 583. Marahiel, M. A,, Lurtz, R., and Kleinkauf, H. (1980). Sixth Znternatiorinl Ferfnetitation Symposium. F.4.3.5 P. London, Ontario, Canada. Margalith, P . , and Pagani, H. (1961). Appl. Microbiol. 9, 325. Marshall, R. Redfield, B., Katz, E . , and Weissbach, H . (1968). Arch Biochem. Biophys. 123, 317. Martin, J. F. (1976). In “Microbiology-1976.” (D. Schlessinger, ed.), pp. 548-552. Amer. Soc. for Microbiology, Washington, D.C. Martin, J. F. (1977a). A h . Biochem. Eng. 6, 105. Martin, J. F. (1977b). Annu. Rev. Mic-robiol. 31, 13. Martin, J. F. (1978). In “Antibiotics and Other Secondary Metabolites. Biosynthesis and Production” (R. Hutter, T. Leisinger, J. Nuesch, and W. Wehrli, eds.), pp. 1 9 3 7 . Academic Press, New York. Martin, J. F . , and Demain, A. L. (1976). Biochetn. Biophys. Res. Cornmun. 71, 1103-1109. Martin, J. F., and Dernain, A. L. (1977a). Can. J . Microbiol. 23, 1334. Martin, J. F., and Dernain, A. L. (1977b). J . Bacteriol. 132, 590. Martin, J. F., and Demain, A. L. (1978). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), pp. 426450. Arnold, London. Martin, J. F., and McDaniel, L. E. (1974). Deu. lnd. Miaobiol. 15, 324337. Martin, J. F., and McDaniel, L. E. (1975). Bintechnol. Bioeng. 17, 925-938. Martin, J. F., Liras, P . , and Deinain, A. L. (1978). Biochem. Biophys. Res. Conmiun. 83, 822. Martin, J . F., Gil, J. A., Naharro, G . Liras, P., and Villanueva, J . R . (1979a). In “Genetics of Industrial Microorganisms” (0.K. Sebek and A. I. Laskin, eds.), pp. 205-209. Amer. Soc. for Microbiology, Washington, D.C. Martin, J . F . , Naharro, G., Liras, P., and Villanueva, J. R. (1979b). J. Antibiot. 32, 600406. Martinelli, E . , Antonini, P., Cricchio, R . , Lancini, C . , and White, R. J. (1978).J. Antibiot. 31, 949-9,51.

Martinez-Molina, E., and Olivares, J. (1979). Can. J. Miaobiol. 25, 1108. Masamune, S., Bates, G. S., and Corcoran, J. W. (1977). Angew. Cheni. 16, 585406. Masurekar, P. S., and Dernain, A. L. (1972). Can. J. Microbiol. 18, 1045-1048. Masurekar, P. S., and Deinain, A. L. (1974a). A p p l . Mic-robiol. 28, 265. Masurekar, P . S., and Demain, A. L. (1974b). Antinticrob. Agents Chen~other.6 , 366. Matern, U.,Brillinger, G. U., and Pape, H . (1973). Arch. Mikrobiol. 88, 3 7 4 8 . Matteo, C. C., Glade, M., Tanaka, A , , Piret, J., and Deniain, A. L. (1975). Biotechnol. Bioeng. 17, 129-143. Maxon, W. D., and Chen, J. W. (1966). J . Fw-ment. Technol. 44, 255-263. Mehta, R. J., Nash, C. H . , Speth, J. L., and Oh, Y. K. (1980). Deu. lnd. Microhiol. 21, 227. Merrick, M. J. (1976). J. Gen. Miaobiol. 96, 299. Mertz, F. P., and Doolin, L. E. (1973). Can. J . Microbiol. 19, 263. Michalic, J., Czerska, W. E . , Switalski, L., and Bojanowska, K. R. (1975). Antimicrob. Agents Chetitother. 8, 526. Micheli, M. R. (1980). Microbiologicn 3, 4 3 4 8 . Mikulik, K., Karnetova, J., Quyen, N., Blurnauerova, M., Komersova, I . , and Vanek, 2. (1971). J. Antihiot. 24, 801409. Miller, A. L., and Walker, J. B. (1969). J . Bactwiol. 99, 401. Miller, A. L., and Walker, J. B. (1970~).J. Bucteriol. 104, 8. Miller, A. L., and Walker, J. B. (1970b). J . Bacteriol. 104, 8.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

111

Miller, J. H., and Reznikoff, W. S. (1978). “The Operon.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Miyamura, S., Koizumi, K., and Nakagawa, Y. (1979). J . Antibiot. 32, 1217. Muth, W. L., and Nash, C. H. (1975). A n t i r n i ~ ~ oAgents b. Chernofhw. 8,321. Nakano, H., Tomita, F., and Suzuki, T. (1974). Agric. Biol. Chem. 38, 2505. Nakano, H., Tomita, F., Yamaguchi, K., Nagashima, K., and Suzuki, T. (1977). Biotechnol. Bioeng. 29, 1009. Nakano, N. M., Ozawa, K., and Ogawara, H. (1980a). J . BactLwiol. 143, 1501. Nakano, N. M., Ozawak, K., and Ogawara, H. (1980b). FEMS Microbiol. Lett. 9, 111. Nakao, Y . , Suzuki, M., Kuno, M . , and Maejinia, K. (1973). Agric. Biol. Chem. 37, 1223. Nakatsukasa, W. M., and Mabe, J. A. (1978). J. Antibiot. 31, 805. Nara, T. (1979). Annu. Rep. Fernient. Proc. 2, 223. Nester, E. W., and Montoya, A. (1976). J . Boctwiol. 126, 699. Nester, E. W., Dale, B., Montoya, and Vold, B. (1974). Biochiin. Biophiys. Acta 361, 59. Nierlieh, D. P. (1978). Annu. Rev. Microbiol. 32, 393432. Nomi, R., and Nimi, 0. (1969). Agric. Biol. Chern. 33, 1459. Nomi, R., Nimi, O., and Kado, T. (1968). Agric. B i d . Chern. 32, 1256. Nyri, L., Lengyl, Z. L., and Erdelyi, A. (1963). J. Antihiot. Ser. A 16, 80. Ochi, K., and Katz, K. (1978). J. Antihiot. 31, 1143. Ogawara, H., and Nozaki, S. (1977). J. Antibiot. 30, 337. Ogawara, H., and Horikawa, S. (198Oa). Antimicroh. Agents Chernofher. 17, 1. Ogawara, H., and Horikawa, S. (1980b). J. Antibiot. 32, 1328-1335. Ohinan, D. E., Cryz, S. J , , and Iglewski, B. H. (1980). J . Bactwiol. 142, 836. Okazaki, H . , Oho, H . , Yainada, K., Beppu, T., and Ariina, K. (1973). Agric. B i d . Che7n. 37, 2319. Okazaki, H . , Bcppu, T., and Ariina, K . (1974). Agric. B i d . Cheni. 38, 1455-1461. Omura, S. Tanaka, H., Okada, Y . , and Marumo, H. (1976). J . Chem. Soc. C h e m Connn. 320-321. Omura, S . , Iwasaki, H., and Kitano, C. (1979). J . Antihiot. 32, 1058. Ortman, R . , Matern, Y . , Grisebach, H., Stadler, P., Siniiwell, P. V., and Paulsen, H. Dur. J. Hiochem. 43, 265. Otsuka, I I . , Quigley, F. R., Groger, D., Anderson, J. A., and Floss, H. G . (1980). Planfa A4etl. 40, 109. Oxford, A. E., Raistrick, H., and Simonart, P. (1935). Biochem. J . 29, 1102. Papc, H . , and Brillinger, G . U. (1973). Arch. Alikrohiol. 88, 25. Pass, L., and Raczynska-Bojanowska, K. (l969a). Acta Biochiin. Pol. 15, 355. Pass, L., and Raczynska-Bojanowska, K. (196%). Acta Biochini. Pol. 16, 297311. Pass, L., Styczynska, D., and Bojanowska, R. K. (1974). Acta. Biochiin. Pol. 21, 213. Pastan, I., and Adhya, S. (1976). Bacterial. Rev. 40, 527-551. Patel, N . , Pierson, D. L., and Jensen, R. A. (1974). ,/. B i d . Che7n. 252, 5839. Paulus, H. (1967). In “Antibiotics” (U. Gottlieb and P. D. Shaw, eds.), Vol. 2, pp, 254-267. Springer-Verlag, Berlin and New York. Peberdy, J. F. (1980). Enzyme Microh. Technol. 2, 23. Pensikova, G. A., and Levitov, M . M . (J970). Biologi!yu 39, 337. Petroski, R. J., and Kelleher, W. J. (1977). FEBS Lett. 83, 55-57. Piggot, P. J.. and Coote, J. 6. (1976). Bactwiol.Eel;. 40, 908-962. Pirt, S. J. (1976). Deo. Intl. Mic~ohiol.17, 1. Pirt, S. J . , and Righelato, R. C. (1967). A p p l . hlic~ohiol.15, 1284. Piwowarski, J. M . , and Shaw, P. I).(1979). Antimicrob. Agents Chetnothcr. 16, 176. Ph, L. C . (1971). Riochim. Biophys. Actu 242, ,541,

I12

VEDPAL SINGH MALIK

Pogell, B. M. (1975). Methods Enzymol. 43, 508. Polsinelli, kf., Albertinc, A,, Cassani, G ., and Ciferri. 0.(19&5).J . Cen. Mimobiol. 39, 239. Prusiner, S . , and Stadtman, E. R . (1973).“The Enzymes of Glutaminc Metabolism.” Academic Press, New York. Puc, A , , and Socic, H . (1977). Eur. J . Appl. Microhiol. 4, 283. Purich. D. L., and Frotnnr, H. J. (1973).J . R i d . Chein 248, 461. Queener. S. W., Sehek, 0. K., and Vezina, C. (1978). Annu. Heo. Alicrobiol. 32, 593. Raczynska, K. B., Ruczdjz, Z.. Rafalski, A,. and Sawnor, D. K. (1973). Antimi~roh.Agents Chcinotlrer. 3, 162. Rafalski, A . , Raczynska, K. R. (1972). Acta Biochini. Pol. 19, 71. Rafalski, A,, and Raczynska, K. B. (1973). Acta Microhid. Pol. 22, 87. Ragan, C. & I . . and Vining, L. C. (1978). Can. J . Microhiol. 24, 1012. Raistrick, H. (1931). Philos. Trans. R. Sor. London S w B 220, 1. Kaistrick, H . , and Clark, A. B. (1919). BZ(~cheiii.J . 13, 329. Ramsev, H . I I . , Hussain, S. M., and Williams, R. P. (1973). Abstr. Annu. Meet. Am. Soc. Microbial. P239, p. 180. Redshaw, P. A., McCann, P . A,, Pentdla, M. A,, and Pogel, B. M . (1979). / . Hacteriol. 137, 891. Richinonrl, M . H . , and Sykes, H. B. (1973). Ada. hfiflfJhiol. Physiol. 9. Riley, XI., antl Anilionis, A . (1978). Annu. Reo. Microbiol. 32, 519-560. Kolhers, J. E . , Robertson, L. W., Hornemarrn, K . M.,Jindra, A , , and Floss, H. C. (1972).J . nnctmioi.112, 791. Roberts, J. W. (1976). In “RNA Polymerase” (R. Losick, ed.), pp. 247-271. Chamberlin, New York. Roeder, 6. S.. and Fink, G. R. (1980). Cell 21, 239. Koetler, 6. S., Farabaugh, P. J . , Chaleff, D. T., and Fink, G . R. (1980). Science 209, 1375. Rogers, T. O., and Birnbaum, J. (1974). Antimicrob. Agents Chemotlzer. 5, 121-132. Rogolskv, M.,antl Nakamura, H. T. (1974).J . Ractcriol. 119, 57-61, Roland, I . , Froyshuv, O., Laland, S. G. (1977). F E B S Lett. 84, 22-24. Rnlinson, G. N. (1979). J . Antimicroh. Chenmther. 5(1), 7-14. Roirrano, A. H.,and Margiotta, E. (1980). In Second Confrrence on Genetics and Molecular Biolog!y of Industrial 1Microcwgunisni.s. Bloomington, Indiana. Rose, A. H. (1980). “Economic Microbiology.” Academic Press, New York. Rosenberg, M.,and Court, D. (1980). Annu. Reo. Genet. 13, 319. Rossi, A,, and Chrcoran, J. W. (1973). Biocheni. Biophys. Res. Coininun. 50, 597. Roszkowski, J. (1972).Acta Microbiol. Pol. Ser. B . 4(1), 9-22. Ruczaj, Z . , Ostrowska, K. B., Sawmor, K. D., arid Raczynska, B. K. (1972). Acta M i ~ ~ ~ b i o l . Pol. 4, 201. Rudd, H . A. M.,and Hopwood, D. A. (1979). J . Gen. Microbiol. 114, 3543. Hudd, B. A. M . , and Hopwood, D. A. (1980).J . Gen. Microbiol. 119, 333. Ruhland, W. (1958).“Encychpedid of Plant Physiology,” Vol. X. Springer-Verlag, Berlin and New York. Sadoff, H . L. (1972). I n “Spores V” (H. 0. Campbell, ed.), pp. 157-166. Ainer. Soc. for hlicrohinlogy, Washington, U.C. Saltero, F. V., and Johnson, M . J. (1953). A p p l . Microbiol. 1, 52-57. Sanchez, S., Paniagua, L.. Mateos, R. C., Lara F., and Mora, J. (1980). Int. F m i e n t . Synip., 6th Tmonto. Sankaran, L., and Pogell, B. M. (1973). Nature (London) New B i d . 245, 257-260. Sankaran. I,., and Pugell, €3. M .(1975). Antimicroh. Agents Cheniother. 8, 721.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

113

Sarkar, N . , and Pauhs, H. (1972). Nafure (London) New B i d . 239, 228-230. Sarkar, N . , Langley, D., and Paulus, H. (1977). Proc. W a d . Acad. Sci. U.S.A. 74, 1478. Satoh, A,, Ogawa, H . , and Satomura, Y. (1976). Agric. B i d . Chein. 40, 191-196. Sawada, Y., Baldwin, J. E., Singh, P. D., Solomon, N . A., and Demain, A. L. (1980a). Antiinieroh. Agents Chemother. 18, 465. Sawada, Y., Hunt, N. A . , and Demain, A. L. (l980b). J . Antibiot. 32, 1303. Sawada, Y . , Solomon, N . A,, and Demain, A. L. (1980~).Biotechnol. Lett. 2, 43. Sawai, T., Takahashi, I., and Yamagishi, S . (1980). Antimimob. Agents Chemother. 13, 910999. Sawula, R. W., and Crawford, I. P. (1973).J. B i d . Chem. 248, 3573. Schaeffer, P. (1969). Bacteriol. Reu. 33, 48-71. Scherer, S., and Davis, R. W. (1980). Science 209, 1380. Schrempf, H . , and Goebel, W. (1979). In “Plasmids of Medical Environmental and Commercial Importance” (K. N . Timmis and A. Puhler, eds.). Elsevier, Amsterdam. Schwartz, J. L., and Schwartz, S. P. (1979). Antimic~ob.Agents Chemother-. 5, 123. Seddon, B., and Fynn, G. H . (1973). J. Gen. Mimobiol. 74, 305. Secldon, B., and Mandis, S. (1978). Biochem. Soc. Trans. 6, 412. Sermonti, G., Petris, A , , Micheli, M., and Lanfaloni, L. (1977).J . Gen. Microbiol. 100, 347. Sermonti, G . , Petris, A , , Micheli, M., and Lanfaloni, L. (1978). Mol. Gen. Genet. 164, 99. Sermonti, G., Lanfaloni, L., and Micheli, M. R. (1980). Mol. Gen. Genet. 177, 453. Setlow, P. (1973). Riochem. Biophys. Res. Coininun. 52, 365372. Shaw, P. D., and Piwowarski, J. (1977).J . Anfibiut. 30, 404. Shaw, W. V. (1979). Ann. N . Y . h a d . Sci. 182, 234. Shaw, W. V., and Hopwood, D. A. (1976).J . Gen. Microbiol. 94, 159-166. Shaw, W. V., Packman, L. C., Burleigh, B. D., Dell, A . , Morris, H. R., and Hartley, B. S. (1979). Nature (London) 282, 870. Silaeva, S. A , , Glazer, V. M . , Shestakov, S. V., and Prokofiev, M. A. (1965). Biokhirniya 30, 947-955. Sims, A. P., Toone, J., and Box, V. (1974). (1974). J . Gen. Microbiol. 80, 485. Simuth, J . , Hudec, J . , Chau, H. T., Danyi, O., andzelinka, J . (1979).J . Antibiot. 32, 53-58. Sjoland, S., and Gatenbeck, S. (1966). Acta Chem. Scand. 20, 1053. Smith, C. G., and Hinman, J . W. (1963). Prog. Ind. Microbiol. 4, 137-163. Smith, R. L., Bungay, H. R., and Pittenger, R. C. (1962). Appl. Mierobiol. 10, 293. Snoke, J. E . , and Cornell, N . (1965). J. Bacteriol. 89, 415. Soltero, F. V., and Johnson, M. J. (1953). Appl. Microhiol. 1, 5257. Soltero, F. V., arid Johnson, M. J. (1954). Appl. Microbiol. 2, 41. Spalla, C., Filippini, S., and Grein, D. (1978). Folia Mimobiol. 23, 505. Spizek, J., Malek, I., Suchy, J., Voridracek, M., and Vanek, Z. (1965). Folia Microbiol. 10, 263-266. Start, W. M., Wilgus, R. M., and Dubus, R. (1980). Deu. 2nd. Microbiol. 21, 77-89. Stephens, J. C . , Artz, S. W . , and Ames, B. N. (1975). Proc. Natl. Acad. Sci. U . S . A . 72, 4389-93. Stewart, W. W. (1971). Nature (London) 229, 174. Stinchcomh, D. T., Thomas, M., Kelley, J., Selker, J., and Davis, R. W. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 4559. Stirling, I., and Elson, S. W. (1979).J . Antibiot. 32, 1125. Stoops, J. K., and Wakil, S. J. (1980). Proc. Natl. Acad. Scd. U.S.A. 77, 4544. Streicher, S. L., and Tyler, B. (1980). Proc. Natl. Acad. Sci. U.S.A. 78, 229. Struhl, K . , and Davis, R. W. (1980). J . Mol. Biol. 136, 1309.

114

VEDPAL SINGH MALIK

Struhl, K., Davis, R. W., and Fink, G. R. (l979a). Nature (London)279, 78. Struhl, K., Stinchcomb, D. T., Scherer, S., and Davis, R. W. (1979b). Proc. Natl. Accitl. Sci. U.S.A. 76, 1035. Struhl, K., Stinchcomb, D. T., and Davis, R. W. (1980). J . hlol. Biol. 136, 291. St. John, T. P., arid Davis, R. W. (1979). Cell 16, 443. Suarez, J . E., and Chatcr, K. F. (1980). Nature {London) 286, 527. Summers, A. 0. (1978). Annu. Reo. Microhiol. 32, 637. Suter, M . , Hutter, R., and Leisinger, T. (1978). I n “Genetics of Actinomycetales” (E. Freerksen, I. Tarnok, and J. E. Thumin. ed Fischer-Verlag, Stuttgart. Snznki. M . , Kuno, M., Maejima. K., and Nakao, Y. (1974). Agric. Riol. Chem. 38, 667. Swain, T. (1977). Annu. Ruu. Plant Pluysiol. 28, 479. Switzrr, R. L. (1977). Annu. Reo. Microbiol. 31, 135. Szeszak, F., and Szabo, G. (1973). Acttr B i d . Acad. Sci. Ifung. 24, 11-17. Tabcr. W. A., arid Vining, L. C. (1963). Can. J. Micrubiol. 9, 1. Tadrew, P. L., and Johnson, M. J . (1958). J . Bactwiol. 76, 400. Takeda Chemical lntlnstries Ltd. Japan (1975). Patcnt JA-110723. Takeda Chemical Industries, Ltd. Japan (1975). Patent J.S. 109680. ’rakeda, K., Aihara, K . , Furumai, T., and Ito, Y. (1979). /. Antibiot. 32, 18-28. Takeda, K., Okuno, S., Ohashi, Y., and Fufiimai, T. (197y78a). J . Antihiot. 31, 1031-1038. Takeda, K., Okuno, S., Ohashi, Y., and Fufumai, T. (197813). J . Antibiot. 31, 1039-1045. J . Antibiot. 31, 1023-1030. Takeda, K., Okuno, S., Ohashi, Y . , and Fufumai, T. (1978~). Tamnra, A., ’I‘akeda, I., Naruto, S . , and Yoshimura, Y. (1971). J . Antihiot. 24, 270. Tatum, E. L., and Gross, S. R. (1954). Proc. Natl. A c n d Sci. U . S . A . 40, 271. Tereshin, I . M. (1976). Poyene Antibiotics, Present and future. Tokyo, University of’rokyo. 144 PP. Thomas, D. Y . , and James, A. P. (1980).J . Bactevioiol. 143, 1179. Thompson, J . M., Ward, J. M., and Hopwood, 1). A. (1980). Nature (London) 286, 525. Tomasz, A. (1979). Annu. Reo. Mic-robiol. 33, 113. Toropova, E. G., Egorov, N. S., and Suchkova, L. A. (1973). Antibiotiki (Moscow) 18, 587. Treichler, H. J., Licrsch, M., Nuesch, J . , and Dobeli, 14. (1979). In “Genetics of Industrial Microorganisms” (0.K . Sebek and A. L. Laskin, eds.), pp. 97-104. Amer. Soc. for Microbiology, Washington. D.C. Tronick, S. R . , Ciardi, J. E., and Stadtman, E. R. (1973). I . Bacteriol. 115, 858. Trowsdale, J.. Shiflett, M., and Hoch, J. A. (1878). Nature (London) 272, 179-181. Tsuji, N., Kohayaski, M . , Terui, Y . , and Tori, K. (1976). Tetrahedron 32, 2207. Turnbough, C. L., j r . , and Switzer, R. L. (1975). J . Bncteriol. 121, 115. Turner, W. B. (1971). “Fungal Metabolites.” Academic Press, New York. Turner, W. B. (1973). I n “Actinomycetalcs: Characteristics and Practical Importance.” The Society for Applied Bacteriology Symp. Ser No. 2.(G. Sykes and F. A. Kinner, eds.), Academic Press, New York. Tylcr, B. (1978). Annu. Rev. Microbiol. 47, 1127-1162. Udvardy, E. N. (1980). Process. Biochem. 15, 5. Umbarger, H. E. (1978). Arrnu. Rev. Biochem. 47, 533. Umezawa, H. (1967). “Index of Antibiotics from Actinornycetes.”Univ. of Tokyo Press, Tokyo. Unowsky, J., and Hoppe, D. C. (1978). J . Antibiot. 31, 662. Vanek, Z.(1958). Folia B i d . 4, 100. Vanek, Z., and Hostalek, 2. (1972). Postep. H i g . M e d . Dows w . 26, 445. Vanek, Z., and Mukulik, K. (1978). Folia Miwobiol. 23, 309. Vanek, Z., Cudlin, J, Blumauerova, M., and Hostalek, Z. (1971). F d i u Microbiol. 16, 225. Vdsdntha, N . , and Freese, E. (1980). 1. Bacteriol. 144, 1119.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

115

Vining, L. C . , and Taber, W. A. (1979). Econ. Microbial. 3, 389. Vining, L. C. (1979). Adu. A p p l . Microbiol. 25, 147. Vining, L. C., and Wright, J. C. (1977). I n “Biosynthesis, A Specialist Periodical Report” Bd. 5, S. 240. Chemical Society, London. Vining, L. C . , Malik, V. S., and Westlake, D. W. S. (1968). Lhydia 31, 355. Vitkovic, L., and Read, E. (1880). A m . Soc. Microbiol. Annu. Meet., Miami. Vitkovic, L., and Sadoff, H . L. (1977a). J. Bacteriol. 131, 891. Vitkovic, L., and Sadoff, H . L. (1977b). J. Bacteriol. 131, 897. Wagman, G . H., and Weinstein, M. J. (1980). Annu. Rev. Microbial. 34, 537. Waksman, S. A. (1943). J. Bacteriol. 46, 299. Waksman, S. A. (1956). G . Microbiol. 2, 1. Walker, J. B. (1964). J. Biol. Chein. 239, 578-581. Walker, J. B. (1974). J. Biol. Chem. 249, 2397. Walker, J. B. (1975). Methods Enzymol. 43, 451. Walker, J. B. (1978). In “Cyclitols and phosphonositides” (W. Wells and F. Eisenberg, Jr., eds.), pp. 423438. Academic Press, New York. . 105-113. Walker, J. B. (1980). Deo. Ind. M i ~ r o b i o l21, Walker, J. B., and Hnilica, V. S. (1964). Biochim. Biophys. Acta 89, 473. Walker, J. B., and Walker, M. S. (1967). Biochemistry 6, 38213829. Walker, M. S., and Walker, J. B. (1971). J. Biol. Chem. 246, 7034. Walker, M . S., and Walker, J. B. (1964). Biochiin. Biophys. Actu 193, 201. Wallace, R. J., Vance, P., Weissfeld, A., and Martin, R. R. (1978). Antimimob. Agents Chernother. 14, 704-709. Watanabe, S., and Tanaka, K. (1976). Biochem. Biophys. Res. Cornmun. 72, 522. Wedler, F. C., and Hoffman, F. M . (1974). Biochemistry 13, 3207. Weinberg, D. (1978). FoZia Microbiol. 23,496. Weinberg, E. D. (1970). A h . Microbiol. Ph!ysiol. 4, 1. Weinberg, E. D. (1971). Perspect. Biol. Med. 14, 565. Weinberg, E. D. (1974). Deu. Ind. Microbiol. 15, 70. Weinberg, E. D., and Tonnis, S. M. (1967). Can. J . Microbiol. 13, 614-615. Weisblum, B., and Graham, M. (1979). J. Bacteriol. 137, 635. Weisblum, G., Holder, S. B., and Halling, S. M. (1979). J. Bacteriol. 138, 990. Whitaker, A. (1980). Process. Biochem. 15, 10. Wickner, W. (1980). Science 210, 861. Wieland, 0. H., Hartmann, V., and Siess, E. A. (1972). FEBS Lett. 27, 240. Wilkerson, S. G., Nash, C. H . , and Queener, S. W. (1978). Int. Symp. Genet. Ind. Micromg,, 3rd, Madison (Abstr.). Wilson, D. B. (1978). Annu. Reu. Biochem. 47, 933. Woodruff, H. B. (1966). Soc. Gen. Microbiol. Symp. 16, 22. Woodruff, H. B. (1980). Science 210, 311. Woyczikowska, B., Pass, L., Girdwoyn, M., and Raczynska, K. G. (1973). Actu Biochirn. Pol. 20, 285. Wright, M. H., and Hopwood, D. A. (1977). J. Gen. Microbiol. 102, 417. Yanagimoto, M . , and Terui, G . (197la). J . Ferment. Technol. 49, 604. Yanagimoto, M., and Terui, G . (1971b). J. Ferment. Technol. 49, 611. Zaidenzaig, Y., Fitton, J. E., Packman, L. C., and Shaw, W. V. (1979). Eur. J. Biochem. 100, 609. Zalkin, 11. (1973). Adu. Enzymol. 38, 1. Zeeck, A., Zahner, H. , and Mardin, M. (1974). Justus Liegigs Ann. Chem. 1100-1125.