Antibiotic Production

Antibiotic Production P Masurekar, Rutgers University, New Brunswick, NJ, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Discovery of Antibiotics Yield Improvement

Glossary factorial design An experimental design based on consideration of the effects of all variables. fermentor A special tank used for production of microbial products. levels Concentrations of variables. mutagen A physical or chemical agent that reacts with DNA and causes chemical modifications.

Abbreviations AP CCD DAOC DHFR DO EES EMS FCC HTS HTST ME MRSA

apurinic/apyrimidinic central composite design deacetoxycephalosporin C dihydrofolate reductase dissolved oxygen ethyl ethanesulfonate ethyl methanesulfonate face-centered cube high-throughput screening high temperature short time sterilization main effect methicillin-resistant S. aureus

Defining Statement Antibiotics have been our major weapon against infectious diseases. This article describes the general principles involved in the discovery and the development of antibiotics. Examples are used to illustrate these principles. In addition, the importance of this endeavor is discussed.

Introduction One of the earlier definitions of an antibiotic came from Waksman, who described it as ‘‘a chemical substance derived from microorganisms, which has the capacity of

174

Engineering Aspects of Process Development and Scale-Up Conclusions Further Reading

mutation The chemical modification caused by a mutagen, which does not result in the death of the cell but changes phenotypic properties of the cell. This change is called mutation. pathogen A microorganism that while growing in a host causes morbidity or mortality of the host. strain improvement Genetic modification of the producer microorganism, which results in improvement in the quality and/or the quantity of the compound produced.

MOP MMS MES MOPS NMU NTG/ MNNG OA OTR PEG UV VRE

methoxypsoralen Methyl methanesulfonate N-morpholino-ethanesulphonic acid N-morpholino-propanesulphonic acid N-methyl-nitrosourethane N-methyl-N9-nitro-N-nitrosoguanidine orthogonal arrays oxygen transfer rate polyethylene glycol ultraviolet vancomycin-resistant enterococci

inhibiting growth, and even destroying other microorganisms in dilute solution.’’ This definition was recently updated as follows, ‘‘Antibiotics are molecules that stop microbes, both bacteria and fungi, from growing (‘static’ antibiotics) or kill them outright (‘cidal’ antibiotics).’’ To accomplish this, these compounds inhibit many of the cellular functions critical for its survival. These include the synthesis of cell wall, protein, DNA, RNA, and lipids. Era of antibiotics began with the discovery and subsequent introduction of penicillin into therapy in the 1930s. The need for the treatment of wounded soldiers in the Second World War resulted in significant commitment of resources for the development and production of penicillin. The miraculous curing ability of this antibiotic initiated worldwide search

Applied Microbiology: Industrial | Antibiotic Production

for new antibiotics, especially to treat tuberculosis. There was no cure for this disease, which caused substantial morbidity and mortality. Waksman discovered streptomycin in 1944, which was produced by Streptomyces. It was active against Mycobacterium tuberculosis, the causative agent of the disease. With this discovery began the search for antibiotics produced by this genus and, indeed, the efforts were well rewarded. Of the 11 900 natural product antibiotics discovered through 1994, approximately 55% were produced by Streptomyces spp.; filamentous fungi produced 22%, nonStreptomyces actinomycetes 11% and other bacteria 12%. A mathematical analysis of the discovery trend of antibiotics

175

from Streptomyces predicted that this genus is capable of producing 150 000 antibiotics. While it is possible to argue against this prediction, the fact remains that only a fraction of the total number of antibiotics synthesized in nature have been isolated. The antibiotics isolated so far belong to a number of chemical classes. Some examples are listed in Table 1. All of the structural classes of antibiotics mentioned in this table, except for fluoroquinolones and the last three, are natural products. As mentioned earlier, the antibiotics target a range of functions critical for the survival of the cell, and this wide range of chemical structures makes it

Table 1 Structural classes of antibiotics Structural classes and subclasses

Structural or biological basis

Examples

-Lactam 1. Penams 2. Penems 3. Cephems 4. Carbapenems 5. Monolactams

Azetidinone attached to pentacyclic or hexacyclic ring Saturated pentacyclic ring Unsaturated pentacyclic ring Unsaturated hexacyclic ring Sulfur in pentacyclic ring replaced with carbon Azetidinone nucleus alone

Penicillin, cephalosporin Ampicillin, amoxicillin Fropenem Cefaclor, cefixime Imipenem, meropenem Aztreonam, carumonam

Aminoglycosides 1. Group I 2. Group II 3. Group III-(A, B, C) 4. Group IV-(A, B) 5. Group V-(A, B, C, D) 6. Group VI-(A, B, C)

Amino sugars Di- or tri-saccharides Streptomycin and derivatives Structure of amino sugar 4,5-di-2-deoxystreptamine 4,6-di-2-deoxystreptamine Presence or absence of amino sugar

-Trehalosamine Streptomycin, bluensomycin Apramycin, hygromycin, garamine Ribostamycin, neomycin Kanamycin, gentamicin Kasugamycin, spectinomycin

Macrolides 1. 14-Membered lactone ring 2. 16-Membered lactone ring 3. Aglycone alterations

Macrocyclic antibiotics As the name indicates

Erythromycin

As the name indicates

Spiramycin

As the name indicates

Azithromycin

Fluoroquinolone 1. Group I, II 2. Groups III, IV

4-Quinolones with or without fluorine atom at position 6 Limited spectrum Broad spectrum Extended spectrum

Nalidixic acid, cinoxacin Norfloxacin, ciprofloxacin, ofloxacin Temafloxacin, levofloxacin

Peptide antibiotics 1. Glycopeptides 2. Lipoglycopeptides 3. Lipopeptides

Peptides Contain sugars Contain sugars and lipids or phospholipids Contain lipid chain

Vancomycin Ramoplanin, teicoplanin Polymyxins, daptomycin

Ansamycins 1. Group I 2. Group II

Aromatic ring with aliphatic side chain Naphthalene-type Benzene-type

Rifamycin No antibiotic known

Tetracyclines

Polycyclic structures of perhydronaphthacene carboxamides Substitution in perhydronaphthacene ring

Tetracycline, chlortetracycline oxytetracycline

Lincosamides

Proline substituted with 49-alkyl chain and a thiooctopyranoside; the whole unit is linked by amide bond

Lincomycin, clindamycin

Chloramphenicol (microbial and synthetic)

p-Nitrophenol connected to propanediol connected to dicholro acetamide

Chloramphenicol

Different classes

Modification of three components

3. Groups V, VI

Different classes

(Continued )

176 Applied Microbiology: Industrial | Antibiotic Production Table 1 (Continued) Structural classes and subclasses

Structural or biological basis

Examples

Benzylpyrimidines 1. Class 1 2. Class 2 3. Class 3

Inhibitors of dihydrofolate reductase (DHFR) Bycyclic derivatives-Anticancer drugs Triazinopyrimiddines-Antiparasitic drugs Benzylpyrimidines-Antibactierial drugs

Methotrexate Pyrimethamine Trimethoprim

Sulfonamides

p-Aminobenzenesulfonamide

Sulfathiazole

Oxazolidinone

Zyvox (linezolide)

Based on the information from Bryskier A (2005) Historical review of antibacterial chemotherapy. In: Bryskier A (ed.) Antimicrobial Agents Antibacterial and Antifungals, 2nd edn., ch. 2, pp. 1–12. Washington, DC: ASM Press.

possible to do so. For example, the -lactams inhibit cell wall synthesis while macrolides affect protein synthesis and fluoroquinolones inhibit DNA synthesis. The market size for antibiotics is quite substantial. It is interesting that after nearly 80 years since their discovery, the -lactams still have the largest share, followed by macrolides and fluoroquinolones (Table 2). In the last two decades, there has been a reduction in the resources committed to finding new antibiotics. There are many reasons for this. Although the total number of antibiotics has grown since the 1970s, the number of those commercialized has decreased. The number of novel structures found also has dwindled. Therefore, most of the effort has been directed at chemically modifying existing antibiotics to increase their potency, spectrum, and stability. For example, today fourth-generation cephalosporins are available for the treatment of pathogens resistant to the older cephalosporin derivatives. Even this approach is becoming less fruitful. Furthermore, the investment needed to bring a drug to market has grown to $300–500 million. In light of the relatively limited market size for antibiotics as compared to some other drugs such as statins, it has been hard to justify the investment in the discovery and the development of new ones. As a result, most of the large pharmaceutical companies have discontinued the search for new antibiotics, leaving it to small biotech companies. However, it does not mean that the former are not interested in new antibiotics; they would rather participate in later phases of clinical trial and do Table 2 Antibiotic market Class of antibiotic

Size of market in $US billion

Cephalosporins Penicillins Macrolides Quinolones Others

9.4 6.7 7 6 6

In 2006 Based on the data from The Global Antibacterial Market: R & D Pipelines, Market Analysis and Competitive Landscape. Arrowhead Publishers’ report (2007).

the marketing of a new one. This fits well with the goals of small biotech companies that lack resources to carry out those functions. With the emergence and wide dissemination of multidrug-resistant pathogenic bacteria, the need for new antibiotics has become urgent. This should make the returns on the investment more attractive and there could be more effort devoted to the discovery of new antibiotics. This trend will be further advanced by the development of new screens with the use of advances made in chemistry, biochemistry, and molecular biology. These screens offer potential for finding antibiotics that attack a specific target. Such antibiotics may have fewer toxic side effects and hence will become more desirable. This article will cover the biological and engineering aspects of discovery and production of antibiotics synthesized by microorganisms. The development of the processes to manufacture antibacterial compounds by either partial or total chemical synthesis is beyond the scope of this article.

Discovery of Antibiotics The antibiotics available today can be classified as natural products, chemically modified natural products (also called semisynthetic antibiotics) and those made by total chemical synthesis. The majority are either natural products or semisynthetic compounds derived from them. Therefore, microorganisms remain today a major focus of the search for new antibiotics. Soils are a rich source of microbial cultures. Other materials that can be used for isolation of microorganisms are plants or plant parts, water, and insects. It should be kept in mind that while microorganisms are well adapted to a variety of ecosystems, some types are specially adapted to specific niches. Furthermore, the classes of microbial cultures obtained from a given environmental sample depend on the isolation method/medium used. The microbial population in a given geographical location can vary with the time of the year, so it is absolutely critical to exploit the samples as much as possible to isolate as wide a variety of cultures. Hunter and colleagues

Applied Microbiology: Industrial | Antibiotic Production

have listed some rules and approaches for isolation of microorganisms for screening. These involve consideration of the types of microorganisms to be isolated, types of ecosystems, types of samples, environmental parameters, and natural substrates in the ecosystem of interest. To isolate microorganisms from environmental samples, these are suspended in water, diluted, and plated on solid media. The choice of the medium depends on the type of microbes to be isolated. A good collection of medium compositions for this purpose is given by Hunter-Cevera and colleagues. From these plates, single colonies are isolated, grown on chosen media, both solid and liquid, and tested in an appropriate screen(s). The choice of medium to grow the isolated cultures for screening is based on the experience of the researcher. If the media to be used for screening purpose are solid, a replica plating technique can be used for higher throughput. The screens used in the early phase of antibiotic discovery were based on either growth inhibition or killing of the target bacteria. This type of screen, also referred to as ‘whole cell screen’, is simple to use and can be carried out either on solid or in a liquid medium. The classic case of discovery of penicillin by Fleming falls into this category. It has been reported that when he went on holiday, he left Petri plates containing a culture of a Staphylococcus sp. uncovered in his laboratory. One of them had a mold colony growing on it and it had inhibited the growth of the bacteria. The fungal culture was subsequently identified as Penicillium notatum and Fleming named the active compound penicillin. This technique was refined by Waksman when he initiated a systematic antibiotic discovery program. After the phenomenal success of the efforts to find new antibiotics in the 1960s and 1970s, the number of structurally novel compounds started to diminish and the need for different types of screens became evident. Many of the active compounds found in the ‘whole cell’ screens turned out to be toxins, so target-based screens were developed. For example, to find antibiotics that act on cell wall targets, mutants were made that had a weakened cell wall. The screen consisted of testing extracts of the fermentation broth against the mutant and the parent. If there was a difference between the zones of inhibition observed with the two, then the active compound(s) produced probably was active against cell wall synthesis. This type of screen required more labor but allowed selection of compounds acting on a desired target. A similar screen can be devised where instead of isolating a sensitive mutant, a resistant mutant is found and used in conjunction with the sensitive parent. Advances in molecular biological techniques allow further refinement of this type of assay. For example, some visual indicator of growth is introduced into the resistant strain and both this strain and the sensitive one are grown on the same plate. The growth differential, if any, can be seen from the response of the indicator. This type of screen reduces the labor required for the use of two strains.

177

As mentioned earlier, the success of whole cell screens to find new antibiotics had diminished. The compounds found were often either toxins or those discovered previously. Therefore, there was a need for more specific type of screens. Taking advantage of the increased understanding of the relevant biochemical pathways, screens were developed to find inhibitors of various steps in these pathways. Operation of these assays was facilitated by the advent of high-throughput screening (HTS). Many exquisitely potent and specific enzyme inhibitors were found in these in vitro screens, yet this approach failed to lead to a clinically useful compound. The reason for this was that the activities isolated in these assays were often ineffective against whole cells or in animal models. The cause for failure may have been that the compounds did not penetrate the cell wall or cell membrane or were degraded, or that efflux systems limited their access to the target. Second, these assays were not robust enough for testing of natural product extracts, consisting of complex mixtures of compounds, and only libraries of pure chemicals could be tested. In spite of the lack of success of in vitro single-target screens, it remained a viable approach to finding new active compounds, but needed to be modified to detect compounds that not only inhibited the target enzyme but also affected growth of the cell. Two examples of such screens are cloning of a target gene under control of a highly regulated promoter and use of ‘antisense’ mRNA. In the first example, the target gene was deleted from the chromosome and a complementary copy expressed from the arabinose promoter on a plasmid. In addition, the chromosomal copy of the arabinose operon, including araB, araA, and araD that code for the arabinose-catabolizing enzymes, was also deleted, thus generating a strain where arabinose regulated only the plasmid-based regulon. The positively regulated araC promoter controls expression of the target gene based on the concentration of the inducer, arabinose. The reduction in the expression of the gene made the strain more sensitive to its inhibitors. Since the screen is a whole cell type, only those compounds that not only affected the target but also killed the cells were detected, thereby overcoming the problems mentioned above. A different approach was used in the second example. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression vector and introduced by transformation into S. aureus. Inserts of the resulting 3117 xylose-sensitive clones were sequenced and the identity of the gene source and fragment orientation was determined by BLAST analysis against the annotated genome sequence of S. aureus MRSA strain N315. From this screen, 2169 clones were found to contain genomic inserts in an antisense orientation, representing 658 unique genes. Induction of antisense resulted in varied growth responses, from reduced growth rate to complete growth inhibition. The clones that

178 Applied Microbiology: Industrial | Antibiotic Production

showed a substantial growth effect were tested for their sensitivity to inhibitors of specific genes under the conditions, where either the antisense was induced or not induced. It was found that these clones were hypersensitive to the inhibitors if the antisense was induced. This characteristic allowed the development of a whole cell screen to look for an antibiotic specific for a target and able to kill the cell. Use of high throughput screens and ultrahigh throughput screens for in vitro assays was mentioned earlier. These were not applicable to microbiological assays because of the complexity of the procedure. However, a microbiological HTS system, made possible because of the progress in robotics and computer technology, was described recently. The system was capable of a potential throughput of 100 000 samples per day. However, the maximum number of assays so far performed is 200 000 per month. The cost of operation was 2–3 US cents per assay, including raw materials, disposal, and waste treatment. This attribute makes the system very attractive for testing large chemical libraries. Thus, beginning with simple assays, where the effect of a putative bioactive extract/solution on the growth of a target microorganism was tested on an agar medium, the search for new antibiotics has now advanced to the use of molecular biological tools. Furthermore, progress in robotics and computer technology is being utilized to improve the probability of finding novel new antibiotics.

Yield Improvement Upon discovery of a new antibiotic activity it was found in almost all cases the desired compound(s) is produced in very low quantities by the original microbial strain isolated from nature. A substantial effort, therefore, is needed to develop an economically viable production process. This involves three different aspects: (1) medium design and optimization, (2) optimization of environmental variables, and (3) genetic manipulation of the producing culture. Medium Design and Optimization Nutritional considerations

The nutritional requirements for growth of the producer microorganism are different from those needed for the production of the desired secondary metabolite. As a result, the medium used for substantial growth is often referred to as the ‘seed medium’ and that used during the production phase is called the ‘production medium’. Both contain carbon source(s), nitrogen source(s), and sources of trace nutrients such as magnesium, sodium, potassium, sulfur, copper, zinc, vitamins and amino acids. They may also contain some buffers, when used in the laboratory. The carbon and nitrogen sources used may be the same in

both the seed and the production medium, but their concentrations can be different. Very often different carbon and nitrogen sources are used in these two types of media. In the early days of fermentation process development, the media contained complex materials of plant and animal origin. Such media are designated as ‘complex’ media. They are usually inexpensive, provide ‘unknown critical’ ingredient, support good growth, and can be developed quickly. Their limitations are that the presence of the crude complex ingredients results in variable performance and pose difficulties in scale-up and may interfere in downstream processing. Fortunately, the increase in our knowledge of biochemistry and microbiology and the availability of fermentors capable of monitoring and controlling various physiological parameters has resulted in the development of media with ‘defined’ ingredients. The use of defined media results in consistent performance, ease of scale-up, a fermentation process amenable to control, and fewer problems in downstream processing. However, these media are costly and often fail to reproduce the high yields obtained with the complex media. Carbon sources provide energy and building blocks for the microorganisms and their choice is very critical for the production of high levels of antibiotics. Commonly used carbon sources include monosaccharides like glucose and fructose, disaccharides like lactose and molasses, and polysaccharides like dextrins, starch and cellulose, polyols like glycerol, oils, and alcohols. Glucose can be obtained in cheap crude form as cerelose and corn-syrup. It is used essentially by all microorganisms. However, it can cause ‘catabolite repression’ of important enzymes of the antibiotic biosynthetic pathway. It was observed that glucose repressed in Nocardia lactamdurans (D(-aminoadipyl)cysteinyl-valine) synthetase and deacetoxycephalosporin C (DAOC) synthase, two enzymes in the biosynthetic pathway of cephamycin C. Second, normally the microorganisms grow at a higher rate on glucose and this is suggested as the cause of inhibition of production. Rapid utilization of glucose often results in a sharp decrease in pH with deleterious effects on production. These problems are usually solved by either using another carbon source or with use of a second slowly utilized carbon source along with glucose. In the former case, the second carbon source does not have the limitations of glucose and in the latter glucose is used first, supporting rapid growth, and the second carbon source supports production. The problem of reduction in pH can be avoided by the use of either inorganic buffers such as sodium or potassium phosphate or organic buffers such as 2-(N-morpholino)-ethanesulphonic acid (MES) or 3-(N-morpholino)-propanesulphonic acid (MOPS) in the laboratory, and in fermenters by employing pH control or slow feeding of glucose. The second important component of the medium is the nitrogen source. These are required for the synthesis of cell

Applied Microbiology: Industrial | Antibiotic Production

components such as proteins, nucleic acids, and cell wall as well as primary and secondary metabolites. Examples of commonly used nitrogen sources are ammonium salts, nitrates, urea, amino acids, protein hydrolysates, and proteins. These crude protein sources can be of plant or animal origin. It should be noted that due to concern about bovine spongiform encephalopathy, the use of animal proteins may cause difficulties with the regulatory agencies. The inorganic nitrogen sources are often used up rapidly, leading to reduction in pH and adverse effects on growth and/or production. Therefore, usually complex organic nitrogen sources are preferred. Amino acids are also used as nitrogen sources, especially in defined media. Of all the amino acids utilized, the most common is monosodium glutamate. Amino acids, in addition to acting as precursors of the metabolites of interest, also can play a regulatory role. The reduction in the production of penicillin G upon addition of L-lysine is an example of amino acid regulation of synthesis of an antibiotic. Phosphate is critical for the biosynthesis of nucleic acids and for energy metabolism. It also can be used as a buffering agent. However, it has been found to exercise a regulatory role in the biosynthesis of many antibiotics. Phosphate concentrations above 10 mmol l1 were found to inhibit the production of antibiotics. The products regulated by phosphate come from different chemical groups such as peptide antibiotics, polyene, macrolides, tetracyclines, and complex antibiotics. The mechanism of phosphate regulation is not fully understood. It has been suggested that ATP concentration is the intracellular mediator of phosphate regulation. Another suggestion is that adenylate energy charge, that is, the ratio of ATP þ 0.5ADP concentrations to that of total adenylate phosphates, is involved in the regulation of synthesis. A recent paper describes the role of PhoR–PhoP in phosphate regulation. Regardless of the mechanism of phosphate regulation, it is important to be aware of the regulatory role of phosphate while designing a production medium. Trace ions, which are important for microbial nutrition, include magnesium, copper, ferrous or ferric, cobalt, molybdenum, manganese, calcium, boron, zinc, sulfate, and chloride. Some of these can also affect the production of antibiotics. Cobalt salts had a positive effect on the production of thienamycin by Streptomyces cattleya. In contrast, the yield of the antifungal compound pneumocandin B0 produced by Glarea lozoyensis was adversely affected by the addition of Zn2þ, Co2þ, Cu2þ, and Ni2þ, so care should be exercised in adding trace minerals to the medium. In the next two sections, the approaches used to take advantage of the understanding of the nutritional requirements for the production of an antibiotic and to design an optimized production medium are described. The first step usually is to study the medium in which the compound of interest was discovered to look for clues in

179

designing a better medium. Next a number of carbon and nitrogen sources, temperatures, and pH values are evaluated. This is normally done with a ‘one variable at a time’ approach, although sometimes statistical methods such as the Plackett–Burman technique are also used. Once the composition of the medium is identified, it is further improved and optimized with the use of an experiment design based on statistical methods. This approach allows testing of multiple variables at the same time. There are many advantages of this approach, the most important being that it saves time and labor. Furthermore, it also can detect interactions between various medium components, which is not possible with the ‘one variable at a time’ approach. For the development of the optimum medium, it is essential that these interactions are taken into account. Classical or ‘one variable at a time’ approach

The development of the medium, either seed or production, begins with the selection of the carbon and nitrogen source to be tested. Often this is based either on clues from the medium used to discover the antibiotic or by choosing one or two members of each of the various types mentioned earlier. These are tested one at a time. For example, to design a medium for the production of rifamycin by Amycolatopsis mediterranei a number of carbon and nitrogen sources were tested. These included monosaccharides, disaccharides, polysaccharides and oils, ammonium salts, soybean meal and peanut meal, and other protein hydrolysates. It was found that glucose and lactose supported the highest yields. However, an increase in their concentration above 25 g l 1 resulted in a decrease in titer. Similarly, ammonium sulfate and ammonium carbonate were good inorganic nitrogen sources and soybean meal and peanut meal were good organic ones. In the case if actinorhodin synthesis by Streptomyces coelicolor, starch up to a concentration of 50 g l 1, was found to support good yields. Glutamate was used as nitrogen source. Phosphate concentrations above 2.5 mmol l1 inhibited the synthesis of actinorhodin. A complex interaction between nitrogen source and phosphate was noted. Although it has been observed that glucose usually is not an ideal carbon source for the production of secondary metabolites, there are examples where it can be used. It was reported that production of novobiocin by Streptomyces niveus was not inhibited by glucose. Interestingly, citrate inhibited the synthesis. In the medium containing both glucose and citrate, the latter was used first and production began after it was exhausted. Glucose supported the production. A careful analysis of the effect of nitrogen source on the production of cephalosporin by Streptomyces clavuligerus was performed. Ammonium salts, nitrates, amino acids, and urea were studied for their effect on growth and antibiotic formation both in the presence and in the absence of glycerol. It was found that amino acids and urea supported

180 Applied Microbiology: Industrial | Antibiotic Production

good growth and antibiotic synthesis. The best yields were observed with L-aspargine as nitrogen source. Good growth was obtained with ammonium salts but no antibiotic production was seen. Antibiotic synthesis was inhibited by 75% when L-asparagine was supplemented with ammonium chloride. The examples given above demonstrate the use of screening and the ‘one variable at time’ approach for carbon and nitrogen sources and phosphate. Statistics-based experimental design

As mentioned earlier, a statistics-based technique, which allows testing of multiple variables at the same time is used in further development and optimization of seed and production media. In the following paragraph, an attempt is made to explain this methodology. There are a number of software packages available for the use of this approach such as JMPR by SAS Institute. These are relatively easy to use. If such software is not available and the experimenter is not familiar with statistics, it is recommended that a statistician be consulted before using this methodology. The most popular technique used for studying the joint effects of several factors is called factorial design. The term ‘joint factor effects’ includes both the main effects (MEs) and the interactions. Since in a production medium there are interactions between various components and the exact mechanisms of these interactions are not understood, this empirical method is used. In application of this approach, the relationship between the variables tested and the final result can be written as y ¼ f ("1,"2, . . ., "k) þ ". Usually, function f is a first- or second-order polynomial. A graphical representation of the solution of this polynomial in three dimension results in a surface lying above the plane representing the factors under consideration. This graphical representation has been referred to as a ‘response surface’ and this empirical model is called the response surface model. If this surface is viewed from the top and all points representing the same yield are connected to produce contour lines of constant response, the resulting graph is called a ‘contour plot’. The response surface and contour plots are extremely useful for analysis of the results of a factorial experiment. Therefore, the terms response surface and factorial design are used to describe this statistical approach. In the following discussion of these methods, general statistical terms are used since these techniques are applicable to a broad range of design and optimization problems. In the context of medium design and optimization, medium components or environmental variables are denoted as ‘factors’ and their concentrations or values are represented as ‘levels’. Factorial design of k factors each at two levels requires 2  2    2 ¼ 2k observations and is referred to as 2k factorial design. To distinguish it from the class of fractional factorial designs, it is also called 2k full factorial

design. Two key characteristics of 2k full factorial design are balance and orthogonality; balance indicates that each factor level appears in the same number of runs and orthogonality defines that for two factors to be considered orthogonal they must appear in all their level combinations in the same number of runs. A design is considered orthogonal if all pairs of factors are orthogonal. The polynomial generated from this design will show the MEs and the interaction effects of the variables. The high and the low values of a variable are denoted by þ and  signs. For example, a high value of variable A will be represented as Aþ and the low value as A–. Let z be the average of observations at high and low values of A. Then the MEs of A are given by MEðAÞ ¼ z ðAþÞ – zðA – Þ

and the interaction effects of variables A and B [INT(A,B)] are given by 1 INTðA; BÞ ¼ fzðB þ AþÞ – zðB – AþÞg 2 1 – fzðB þ A – Þ – zðB – A – Þg 2

This equation can be generalized for k factors as follows: 1 INTðA1 ; A2 ; . . . ; Ak Þ ¼ INTðA1 ; A2 ; . . . ; Ak – 1 Ak þÞ 2 – ðA1 ; A2 ; . . . ; Ak – 1 Ak – Þ

Fundamental principles for factorial effects are hierarchical, effect sparsity, and effect heredity. Hierarchical ordering principle can be described as follows: 1. lower order effects are more likely to be important than higher order effects; 2. effects of the same order are equally likely to be important. The effect sparsity principle means that the number of relatively important effects in a factorial experiment is small. The last principle states that for an interaction to be significant, at least one of its parent factors should be significant. These principles are important to the analysis of the results of a factorial experiment. Simple two-level experiments assume a linear relationship between the levels of the factor and their effect. Very often, this is not true and to determine the curvilinear relation it is necessary to test three levels. As the number of levels and factors to be tested increases the number of experiments needed grows exponentially and the full factorial design becomes less feasible. For example, with six factors the full factorial design will require 64 experiments. Only 6 of the 63 degrees of freedom are used to estimate the MEs, while 15 degrees of freedom are used to estimate two-factor interactions. The remaining 42

Applied Microbiology: Industrial | Antibiotic Production

degrees of freedom are associated with the three-factor and higher interactions. It has been generally observed that in medium design and optimization the three-factor and higher interactions are not important, so it is not necessary to use a full factorial design. The information relevant for the purpose can be obtained by carrying out only a fraction of the full factorial design. These fractional factorial designs, because of their efficiency, are the most popular design type used in the industry. These are designated for k factors and two levels as 2kp designs. For example, 2k1 will be one-half factorial, 2k2 will be one-quarter factorial, and so on. While the fractional factorial designs save labor and time, they do have limitations. One consequence of choosing to run only a fraction of the full factorial design is that it is not possible to separate MEs from some of the interactions. Two or more effects that have this property are called ‘aliases’. The extent of aliasing is used to define the quality of the design. It is designated as ‘resolution level’. The higher the resolution the better is the design because the less restrictive are the assumptions that are required regarding which interactions are negligible to obtain a unique interpretation of the data. A number of designs have been developed based on the theoretical considerations listed in this section. A few of the more popular ones are described below. Central composite design

This design is by far the most popular second-order design used for process optimization. In this type of design, two-level factorial or fractional factorial (resolution V) is combined with 2k axial or star points and nc central points. The resolution V design contributes to estimation of linear terms and two-factor interactions; axial points contribute to estimation of quadratic terms, and central points provide the estimation of pure error. Selection of , the axial distance, and nc allows flexibility in this design. The axial distance is selected based on the nature of the design region, whether it p isffiffiffi spherical or cuboidal. In the spherical region,  is k and in the cuboid region it is 1. In the spherical region, it is most effective to use three to five center runs (nc). In contrast, in the cuboidal region one to two center runs are adequate. In cuboidal design the axial points are on center of the faces and hence it is often referred to as a face-centered cube (FCC). Simplex design

This is another popular design, because it requires fewer experiments to find optimal conditions. Regular simplex is a first-order design, wherein a minimum number of experimental points (in any number of dimensions) are called for. For example, in k dimensions it requires (k þ 1) points. In two dimensions, the regular simplex is an equilateral triangle; in three dimensions it is a tetrahedron,

181

and so on. The design is both efficient and ‘rotatable’. It has been shown that this design without replication is optimal for the estimation of slope in the presence of error. For the present purpose of optimization, however, the additional (and unique) attraction is that it is possible by adding just one further point to complete a new simplex on the face of the original simplex design. Thus to the question ‘where to move’, the answer is ‘into an adjacent simplex’. Only one further observation is needed to complete this new simplex and enable the experimenter to ask that question once more. It may be shown that the direction of the steepest ascent estimated from observations at the vertices of regular simplex will proceed from the center of simplex through that face of the simplex, which is opposite to (does not contain) the point corresponding to the lowest observation. It is important to note that the path taken by a simplex design will turn around the optimum and no more improvement in the results can be seen. To define the optimum more precisely, a second series of simplex experiments can be undertaken where the size of the change in the level of a factor is reduced compared to the first series.

Plackett–Burman design

Often, it is not possible to carry out the number of experimental runs as required even by fractional factorial design and at such times different types of designs are required. The factorial designs in which it is possible to estimate any two factorial effects independently of each other, or they are fully aliased, are referred to as ‘regular design’. Designs that do not possess this property are called ‘nonregular designs’. As mentioned above, these designs are used for the reasons of run size economy or flexibility. The nonregular designs not only require fewer runs but also can accommodate various combinations of factors with different numbers of levels. These designs are built from orthogonal array (OA). Plackett and Burman described a large collection of this type of arrays. These are denoted as OA(N,2k), where N is the number of runs and k ¼ N1 variables. Other characteristics of N are that it is a multiple of 4 but not a power of 2. Therefore, the smallest design is N ¼ 12. Some of the other designs commonly used consist of N ¼ 20, 24, and 28. In the early phase of medium development, a number of ingredients are tested or screened for their usefulness. Plackett–Burman designs are very suitable for this purpose. In a 12-run experiment, the first seven columns of the array represent seven medium components and the last four are dummy variables. The dummy variables are used to measure the error in the experiment, which is then used to determine the significance of the effect of any of the variables tested. The experimental arrays based on the Plackett–Burman design can be generated. This

182 Applied Microbiology: Industrial | Antibiotic Production

design has been very useful in rapidly screening a large number of variables. Box–Behnken design

Medium development and optimization for the production of fermentation products involves studying quantitative variables and this design was developed specifically for such a purpose. It is a class of three-level incomplete factorial designs useful for estimating the coefficients in a second-degree polynomial. As described by the authors their aim was, where possible, to generate second-order rotatable designs. To do so, three levels taken by the variables x1, x2,. . .,xk are coded: –1, 0, and þ1 and it is assumed that the second-degree graduating polynomial fitted by the method of least squares: yˆ ¼ b0 þ

k X i¼1

bixi þ

k X k X

bijxixj

i¼1 j ¼i

One of the characteristics of Box–Behnken design is that it is spherical. It can be imagined that for three variables, all experimental points fall at the center of the edges of a cube. The Box–Behnken design is quite comparable to the central composite design (CCD) in the number of runs required. For example, for three variables the former design will contain 12 þ nc runs and the latter will have 14 þ nc runs. Both the Box–Behnken and the CCDs are popular with experimenters. One of the reasons for their popularity is the run size, which is large enough to provide a comfortable margin for lack of fit but not so large as to involve wasted degrees of freedom or unnecessary experimental expense.

Optimization of Environmental Variables The environment in which a microorganism is grown has significant influence on its metabolism, and to improve the production of a desired antibiotic it is necessary to control it. The environmental variables known to affect the production are pH, temperature, aeration, agitation, and the like. In this section, the effects of pH and temperature are considered. We will consider the effects of the rest later in the article. 1. pH The pH of the seed and production media strongly affects the physiology of the producing culture. It has been observed that low or high pH values have a negative effect on yield. At the laboratory scale, the pH is maintained in the range of 5.5–7.0 with buffers. Popular buffers used are, as mentioned above, MES and MOPS. In the large-size fermenters, sodium or potassium hydroxide or sulfuric acid are used to control the pH. Sometimes ammonia gas is used for this purpose and to act as a nitrogen source. As our

understanding of the process increases, it is possible to control the pH at different values; one optimum for growth and another for production. 2. Temperature Process temperature is one of the most important variables to be controlled for good production. It has been noted that the temperature that is optimum for good growth is not conducive to high production and vice versa. However, sometimes it is not convenient to operate the process at multiple temperatures so a compromise value is selected. For example, in the early development of the process for the production of penicillin, it was found that Penicillium chrysogenum grew well at 30  C, whereas the optimum temperature for production was 20  C. Therefore, 25  C was chosen as a compromise temperature. The importance of these two variables to obtain consistently good productivity at all scales of operation, from laboratory to production, cannot be overstated. As a result, these are optimized at the early phase of process development. Often this is combined with the development of the seed and production media.

Genetic Manipulations This is commonly referred to as strain improvement. Its goals are to improve the quantity and the quality of the antibiotic produced, improve the excretion for an extracellular antibiotic, reduce the sensitivity to product inhibition, shorten the production phase, and make the culture amenable to the use of inexpensive and crude medium ingredients. At the same time, as the optimization of medium and environment is being done, the producer microorganism is being mutagenized to increase the yield. Past experience has been that while the former two approaches do result in higher titers, substantially more improvement has been obtained by mutagenesis. The classical approach involves subjecting the microbial strain to a physical or chemical mutagen and screening the survivors for production of the antibiotic. The goal of the screen can either be a quantitative increase in the yields or a reduction in the impurities. Sometimes it is possible to use a selection method to reduce the number of survivors tested. It has been observed that with an effective mutation treatment, about 1000–1500 colonies have to be screened to find an improved mutant. In recent years, molecular biological techniques have become available and these have been applied to strain improvement. Classical mutagenesis Physical mutagens

These include ultraviolet (UV) light and ionizing radiation like X-rays, -rays, as well as fast neutron exposure.

Applied Microbiology: Industrial | Antibiotic Production

Of these, due to ease of use and safety, UV light is preferentially used. Short-wave UV light (i.e., below 300 nm) is very effective in causing mutations since the bases in DNA strongly absorb in this region. Germicidal lamps which emit light at 254 nm are commonly used. It is important to have the cell suspension in a shallow layer and to keep it agitated because the penetration of UV into an aqueous medium is limited. The mechanism of action of UV irradiation is formation of pyrimidine dimers. These could be between C-5 and C-6 of adjacent thymines or between C-6 of thymine and C-4 of cytosine. The presence of these dimers causes distortion in the structure of DNA, which results in stalling of DNA polymerase and hence of replication. In addition to the mechanisms evolved to prevent interruption or errors during replication, a mechanism specific to repair the damage caused by UV light, which depends on the availability of light with wavelengths greater than 350 nm, has been observed. The activity of this system is referred to as ‘photoreactivation’. Therefore, for successful UV mutagenesis of microbes that possess this system, it is important to keep the samples exposed to UV light in the dark for some time before plating for isolated colonies for screening. The dimers also induce SOS response, which in turn leads to mutation. Long-wave UV light (320–400 nm) by itself is not a very effective mutagen, but in the presence of psoralens or their derivatives like 8-methoxypsoralen (MOP), it can be mutagenic. These compounds on exposure to the longwave UV light form adducts with pyrimidine bases, commonly by addition across 5,6 double bond in thymine. MOP can form adducts at both ends of the molecule (double adduct). When the two bases are located on two different strands of DNA, the double adduct crosslinks the two strands, leading to distortion of a DNA structure. This in turn elicits responses from DNA repair systems, including the SOS response. Ionizing radiation affects DNA both directly and indirectly. It interacts, in addition to DNA, with other molecules present in an aqueous medium. Upon the absorption of the energy, these molecules form reactive species called ‘free radicals’. These in turn react with DNA. The DNA damage caused by ionizing radiation includes damaged bases and to a lesser extent damaged sugars and strand breaks. As mentioned above, due to the difficulties involved in using these mutagens and the safety consideration, these are used very rarely. Today penicillin is produced by many manufacturers all over the world. The first improved penicillin producing mutant, X-1612, was isolated with X-ray irradiation. The strain Q-176, the progenitor of most of the strains used in the world, was obtained at the University of Wisconsin by UV irradiation of mutant X-1612.

183

Chemical mutagens

For strain improvement, these are more commonly used than physical mutagens. They are classified based on the type of modification of DNA caused by them. The chemical reactions include alkylation, deamination, or substitution of bases and intercalation between bases. Alkylating agents react with nucleophilic centers in the bases. Although there are a number of these present in the bases, it seems that there is preference for the positions based on the alkylating agent used. For example, alkyl sulfates react exclusively with nitrogen, whereas Nnitroso compounds react primarily with oxygen in the bases. Alkylalkane sulfonates fall in between the two. Further studies to characterize the effects of alkylating agents led to the following conclusions: 1. Bulky substituents at any position will interfere with replication. Some of these products can undergo rapid depurination and cause mutations via error-prone repair. The third possibility is bypass by polymerase, which can lead to frame-shift mutations. 2. Smaller alkyl derivatives are likely to cause misincorporation. O-alkyl derivatives generated by N-nitroso compounds fall in this category. Two repair mechanisms induced specifically by alkylation of DNA include the N-glycosylase-dependent pathway and the system involving methyltransferase. N-glycosylase removes the alkylated bases and apurinic/apyrimidinic (AP) endonuclease cuts the apurinic strand. This is followed by digestion of the cut strand by exonuclease and the resynthesis of the removed strand by DNA polymerase I. In the case of the latter, the methyltransferase removes the alkyl group from the alkylated bases by transferring these groups to itself. In addition, the alkylation of DNA also induces the error-prone repair mechanism. Some commonly used alkylating agents are listed below. Dialkylsulfates: dimethyl- or diethylsulfates are examples of this group. They preferentially alkylate the N3 position of adenine and the N7 position of guanine. The methylating agents are more effective mutagens than the ethylating ones. The reason may be that the ethylating agents are considerably less reactive. Overall, the sulfates are less effective than the sulfonates. Alkyl alkanesulfonates: these react with the O6 position of guanine in addition to the nitrogen atoms in adenine and guanine mentioned before. Methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and ethyl ethanesulfonate (EES) are commonly used members of this group. It was observed that the alkylation of the oxygen atom results in more effective mutagenesis and that these compounds are liquid and hence easy to use, making them attractive candidates as mutagens. N-nitroso compounds: N-methyl-N9-nitro-Nnitrosoguanidine (NTG or MNNG) is the most used

184 Applied Microbiology: Industrial | Antibiotic Production

mutagen for the improvement in production of antibiotics. There are a number of unique features which make this a very effective mutagen. It was noted that the pH and the temperature can be used to improve the efficiency of mutagenesis. Second, NTG was found to preferentially act on the replicating region. This offered a possibility of isolating different types of mutants depending on the region of DNA being replicated. Other N-nitroso compounds used are N-alkyl-Nnitrosourea, N-methyl-nitrosourethane (NMU), and N-dialkyl-N-nitrosoamines. The nitrosoureas react with the oxygen atom in DNA and that makes them very effective mutagens. Miscellaneous alkylating agents include lactones like -propiolactone, epoxides such as ethylene oxide, N-mustard, S-mustard, and halonitrosoureas. These predominantly act on the nitrogen of adenine and guanine. S-mustards also react on the nitrogen of cytosine. Both N-and S-mustard also crosslink the two strands of DNA. Deaminating agents These compounds remove the amino groups in adenine, guanine, and cytosine. Deamination reactions result in altered bases and lead to mispairing, which if not corrected will cause mutations. The specific repair system, which involves specific glycosylases to identify and remove the deaminated bases, and AP nuclease, which makes a cut next to the position from where the base was removed. The repair DNA polymerase then synthesizes new DNA starting from the cut, using the other strand as a template, thus replacing the modified base with the correct one. Nitrous acid : Nitrous acid (HNO2) causes oxidative deamination. It is simple to use and mutagenesis can be easily controlled. The results are predictable. Therefore, it is one of the most commonly used mutagens. The kinetics of deamination is faster at acidic pH and hence the mutagenic treatment is done at pH 4.5. Nitrous acid is generated in situ by adding NaNO2 solution to an acidic buffer. This compound is also used for in vitro mutagenesis. Intercalating agents Compounds with planar structure can intercalate into DNA, forcing the bases in the strand with the intercalated compound apart and causing slippage between the two strands. During replication an extra base may be added to correct for slippage or a base may be deleted. The mutations resulting from this type of DNA damage are called frame-shift mutations, because they change the triplet reading frame. Some acridines were noted to be mutagenic in some systems but are not very effective in bacteria. A series of acridine derivatives was synthesized at the Institute of Cancer Research (Philadelphia, PA). These are called ICR compounds and are effective mutagens.

Mutagenesis protocols For mutagenesis it is necessary to obtain single-cell propagules of the culture to be mutagenized. The spores are an excellent candidate for the treatment. In the case of single-cell microorganisms vegetative cells can be used. In the case of filamentous microorganisms, which do not sporulate, the mycelium can be homogenized and single-cell fragments can be isolated by a combination of filtration and centrifugation. Alternatively, the mycelium can be exposed to the mutagen and protoplasted to obtain single-cell propagules. Next the appropriate mutagen is chosen by preliminary screening of a few. In these preliminary experiments, simple criteria such as morphology of the survivors can be used. In the second series of experiments, the operating conditions such as concentration of the mutagen, time of exposure, temperature, pH, the type of buffer, and so on, are determined. Often the extent of reduction in viability is used as a criterion to standardize the process. In nonselective screening, the survivors are plated on a growth medium at a number of dilutions and isolated colonies are tested for production. In contrast, in selective screening, a selection agent is added to the plates and the surviving colonies are tested. It has been observed that almost all screening in strain improvement programs is nonselective because of a lack of knowledge about the physiology of the producer culture, biosynthesis of the antibiotic and its regulation. In the last stage, the performance of the putative superior mutant is compared with that of the parent in a statistically designed experiment to confirm the improvement. This protocol has been used over last 60 years to increase the yield of antibiotics from penicillin to daptomycin. Since yields obtained in the process are of great economic importance published information available on this topic is limited. It is true that there are publications on strain improvement, but these do not reflect commercial reality. An exception to this rule is probably the production of penicillin. Maybe this is because initially the production process was developed cooperatively by government laboratories, universities, and industry. For strain development for the production of penicillin many mutagens were used. These included physical mutagens such as UV, X-rays, and -rays, as well as chemical mutagens such as NTG, EMS, ICR-170, and nitrogen mustard, to mention a few. Sometimes the chemical and physical mutagens were used in combination. The sustained effort of more than 50 years has resulted in titers of above 100 g l 1 and has made penicillin more of a bulk chemical. Similarly, the work done on mutagenesis of Acremonium chrysogenum at the University of Wisconsin was also published. In this case, in addition to the number of mutagens, selection agents were also used. These included

Applied Microbiology: Industrial | Antibiotic Production

methionine analogues such as selenomethionine, polyene antibiotics, and heavy metal ions. Yields of more than 30 g l 1 were reported. Tetracycline strain improvement programs were also highly successful and titers of 20 g l 1 were reported nearly 30 years ago. Genetic methods Recombination

Prior to the advent of molecular biological techniques, those used in the classical genetic studies were tried for strain improvement purposes. Early efforts were not successful due to the low recombination frequency in antibiotic-producing strains. For example, in strepomycetes it is less than 106 and in commercially important antibiotic-producing fungi such as Penicillium or Acremonium recombination is not observed often. The difficulties caused by the low frequency were reduced with the use of protoplast fusion. During a mutation program, it has been noted that the improved producers acquire a number of undesirable characteristics such as poor sporulation and slow rate of growth. In such a case, it has been possible to eliminate the unwanted properties by back-crossing the high producers with one of the early mutants. This approach was tried to correct for the slow growth rate, and lack of sporulation in high yielding strain of A. chrysogenum by crossing it with an earlier strain, which had low titer, faster growth rate, and sporulated. One of the recombinant strains grew faster, sporulated, and produced 40% more antibiotic than the high-yielding parent. A similar observation was made when a high-producing P. chrysogenum strain was backcrossed with an earlier strain in the genealogy. A different use of recombination was described for improvement in the production of cephamycin C. Two superior mutants were fused and two further improved producers were found in the recombinant progeny. Transposon mutagenesis

Transposable elements are DNA segments that can move to a new location in DNA molecules by a process that requires neither extensive DNA sequence homology between the elements and the site of insertion nor the rec genes needed for classical homologous crossing over. These elements are diverse in size and functional organization. Despite their apparent diversity, the transposable elements are united by countless common features. Transposition generally requires element-specific proteins termed transposes. These bind to the ends of their respective elements and cause transposition. The transposable elements cause mutation when they insert in a new site; they also alter the pattern of expression of the genes near the insertion site. Transposon mutagenesis has been used to increase the production of daptomycin. A number of transposons were

185

constructed from IS 493. One of them, Tn 5099, was introduced into several streptomycetes by protoplast transformation or conjugation from Escherichia coli. Streptomyces roseosporus was subjected to Tn5099 treatment and it was found that more than half of the transposed mutants produced more than the parent. The transposable elements can also be used to clone a gene. It was noted that in tylosin biosynthesis by Streptomyces fradiae, conversion of macrocin was ratelimiting. By cloning the tylF gene, which codes for macrocin O-methyltransferase, the terminal step in tylosin biosynthesis, production of the antibiotic, was increased at the expense of macrococin. However, the total macrolide yield was not changed. Application of molecular biological methods

Development of these methods opened up an avenue that had not been available for strain improvement. Classical mutagenesis affected more than one target on the chromosome(s) and often resulted in improved mutants, which had some undesirable properties, as mentioned earlier. The development of this methodology, also referred to as ‘recombinant DNA’ technology or ‘genetic engineering’, offered a possibility of affecting only a desired step, that is a rate-limiting step, in the biosynthesis of an antibiotic. This approach is called ‘metabolic engineering’. There are many definitions of metabolic engineering: the one used here is, ‘modification of a rate-limiting step(s) in the production of an antibiotic’. According to this definition, the rate-limiting step can be in the biosynthesis of the antibiotic or in that of one of the precursors. The steps involved in the application of this technology are as follows: (1) identification of the ratelimiting step; (2) isolation of the gene that encodes the enzyme which catalyzes that step; and (3) improving the expression of that gene to eliminate the limitation. In the early development, E. coli, Saccharomyces cerevisiae, and Bacillus subtilis were used as model microorganisms. It was not possible to apply this technique to fungi in the early days, because of the poor efficiency of transformation in fungi. Considerable effort was spent on developing the procedures for effectively protoplasting fungi. Next the methods to introduce DNA into fungal cells had to be developed. These included electroporation, and the use of intact cells exposed to alkali cation like Liþ. It was found that for efficient transformation of protoplasts, addition of polyethylene glycol (PEG) or CaCl2 was needed. For electroporation, these additions were not needed but the transformation efficiency was low. The vectors used in E. coli were modified for use in fungi. The transformed DNA is integrated into the chromosomal DNA, homologously in yeast and nonhomologously in filamentous fungi. A new method called Agrobacterium tumefaciensmediated transformation was developed for transformation in fungi. This system offers advantages of simplicity

186 Applied Microbiology: Industrial | Antibiotic Production

and general applicability. For selection of transformants, resistance to hygromycin, produced by Streptomyces hygroscopicus, is often used. This technique was applied to yield improvement of cephalosporin, produced by A. chrysogenum. The biosynthetic pathway for penicillin/cephalosporin had been studied for a long time and considerable information was available. The biosynthetic genes were isolated and characterized. It had been observed that penicillin N was excreted in the broth during cephalosporin fermentation. The molar ratio between cephalosporin C and penicillin N was 0.3, which suggested that conversion of penicillin N into DAOC was rate-limiting. This step is catalyzed by DAOC synthase encoded by gene cef EF. An additional copy of the gene was introduced into A. chrysogenum strain 394-4, which was used for industrial production of cephalosporin. Hygromycin resistance was the selection marker. A number of transformants were found to make more cephalosporin C and less penicillin N. One of them was found to produce 47 and 15% more antibiotic at laboratory scale and in pilot plant fermentor, respectively, as compared to the untransformed parent. Southern hybridization analysis of the total DNA of the parent and the transformant showed that the transfomant contained an extra copy of cef EF. Furthermore, DAOC synthase activity in the crude extract was found to be doubled. These results showed that there was no one-toone correlation between gene dosage and the yield of antibiotic. It also suggested that there was another step that was rate-limiting. Later it was shown that introduction of an additional copy of gene cef G, which codes for cephalosporin C acetyltransferase, increased the production in a dose-dependent manner, indicating that this was probably the rate-limiting step. Molecular biological techniques can also be used to increase the expression of a positive regulator or to disrupt that of a negative regulator of a biosynthetic pathway. An example of the former is the increase in the production of spiramycin in Streptomyces ambofaciens and of the latter the increase in the nystatin yield by disrupting nysF, a negative regulator in Streptomyces noursei. There are two examples of proprietary methodologies to increase the quantity and quality of the desired product including antibiotics. One of them developed by Maxygen and later licensed to Codexis Inc. (Redwood City, CA) is called ‘Molecular Breeding’. It involves gene shuffling or genome shuffling. The former is useful for improving the properties of a single enzyme and the latter for increasing the yield of a microbial product. Genome shuffling combines the multiparental crossing allowed by DNA shuffling with the recombination of entire genomes. In such a system, when a number of phenotypically selected strains are used many of the progeny show improvement in that particular phenotype. To begin this process, the wild type or the starting strain is mutagenized

to generate clones with improved phenotype (e.g., higher titer than the parent). A few of these are combined, protoplasted and fused, thus resulting in multiparent crosses. A number of progeny from this process are subjected to HTS to select further improved clones, which then are treated in the manner described above. This process was used to increase the production of tylosin by S. fradiae. In two rounds of genome shuffling, the yield was improved 6- to 8-fold. It is important to note that the improved producers isolated by this method in 1 year were as good as or better than those generated by the classical strain improvement program in 20 years. However, there are no reports of use of this method for strain improvement in molds. The second example, a technology termed ‘Precision Engineering’, was developed by Microbia (Ironwood Pharmaceuticals; Cambridge, MA). It is based on combination of metabolic and transcriptional profiling to identify the genes associated with the desired product formation, use of global regulators, and high-throughput genetically selective screening. The transcriptional screening is done with microarrays. They have a library of more than 400 regulators associated with a variety of physiological regulatory pathways from diverse microorganisms. They believe that this approach allows them to address any specific regulatory mechanism directly and results in rapid strain improvement (www.ironwoodpharma.com). This technology was developed for application to fungal strain improvement. These two technologies, probably because of the use of nonspecific screening, have a high probability of success. Lastly, the molecular biological techniques can be used to generate novel antibiotics. This approach has been pursued by Kosan Biosciences Inc. They have modified or deleted various domains of the polyketide synthases involved in the biosynthesis of erythromycin and other macrolides to generate novel compounds with different properties from those of erythromycin. Application of this approach to generate novel antibiotics will certainly increase since the classical method of screening microorganisms for new compounds has not been successful in the last 30 years. As our knowledge of the biosynthetic pathways and their regulation at the molecular level increases, these techniques can be employed to eliminate or modulate the rate-limiting steps. There are examples in the literature of the use of these approaches at the laboratory scale and they certainly will find utility in strain improvement, even if the results are not published.

Engineering Aspects of Process Development and Scale-Up The topics discussed so far dealt with work done at the laboratory scale. However, for manufacture of the

Applied Microbiology: Industrial | Antibiotic Production

antibiotic, the optimized process has to be scaled up to larger sizes, initially to a pilot plant and finally to a production facility. One of the major differences between the laboratory size and the larger scale is that at that level the process is operated in fermenters, which are significantly different from the type of reactors used in the laboratory. As a result, a number of new engineering variables have to be considered. These include aeration, agitation (mixing), rheological nature of the fermentation broth, heat transfer, and sterilization of the medium. It is important to recognize that each of these variables affects the others.

Aeration and Agitation Antibiotics are produced in an aerobic process, that is, the producing microorganism requires oxygen for growth; it is therefore necessary to supply adequate oxygen. The relationship between the oxygen transfer rate (OTR) and the physical parameters is given by the following equation: OTR ¼ KL aðC  – CÞ

where KL is the liquid film mass transfer coefficient, a is the interfacial area between the gas phase and the liquid phase, C is the saturation concentration of oxygen in the liquid, and C is the concentration of oxygen in liquid. This equation indicates that to improve the transfer rate, it is necessary to increase the values of KL, a, and C. In fermentors, the air is sparged at the bottom of the tank and the air bubbles, as they rise to the top, are sheared by multiple impellers, leading to generation of smaller bubbles. These are due to their large interfacial area, critical for good OTR. In addition to shearing air bubbles, agitation is necessary to keep the fermentation broth well mixed to avoid any local limitations of oxygen or of any medium ingredient. Both these requirements, that is shearing air bubbles and pumping of medium have to be considered in impeller design. It has been recognized that the biggest resistance to oxygen transfer from air to liquid medium is that of the liquid film. It was found that KL.a is affected by many operating variables. Cooper and his coworkers showed that, for a vaned disk impeller, volumetric mass-transfer coefficient (Kv) was correlated with the unit power input (Pv) and superficial gas velocity (Vs) as shown by the following two equations: Kv ¼ kPv0:95 and Kv ¼ k9Vs0:67

where k and k9 are constants. In addition to the relationship shown above, subsequent studies have shown it to be proportional to impeller tip speed and to apparent viscosity in nonNewtonian fermentation broths. As indicated by these

187

equations, increases in the power input and in superficial velocity were necessary to obtain higher KLa. However, since there is a limit to the increase in power input and in superficial velocity, the impellers and the fermenters had to be redesigned. The most common impeller type used was the Rushton turbine. It was capable of high shear but did not have high pumping capacity. To increase the pumping capacity, multiple Rushton turbine impellers were used in fermentors from the early 1950s to the early 1980s. However at present, agitators with other design types are available, for example, axial flow hydrofoil impellers. These offer better performance over the Rushton impellers for large-scale viscous, mycelial fermentations. The specific advantages of this type of impeller are as follows: improved oxygen transfer per unit power, lower maximum shear rates, and improved bulk mixing. The last characteristic results in elimination of compartmentalization of flow obtained with multiple Rushton impellers, as well as that of local nutrient limitation and hot spots, and offers better control of pH. It is important to recognize that the relationship between the diameter of the tank to that of the impeller (D/T) can affect the efficiency of aeration. The factors that affect this are aeration rate and the mixer power input. There are many examples of effects of agitation and aeration on the production of antibiotics. The mean hyphal length of P. chrysogenum was affected by the circulation frequency and the energy dissipation around the impeller. There was a direct correlation between the mean hyphal length and titer. In the case of erythromycin production by Saccharopolyspora erythrea, it was found that there was an optimum range of agitation of 350–1000 rpm. Any increase above this range reduced the production. Similar observations were also made in the case of tocamycin produced by Streptomyces chrestomyceticus; the yields were adversely affected by tip velocities higher than 275 cm s 1. As more and more fermentation processes were developed, it became clear that it is not only the rate of oxygen transfer but also the concentration of dissolved oxygen (DO) that has an effect on production. It was found that there was a critical concentration of DO, below which production was adversely affected. Therefore, it is imperative to maintain it above the critical level for the duration of the run. With the advent of sterilizable DO probes, it became possible to measure this parameter during fermentation. However, it is important to determine the location of the DO probe carefully. The reason is that, due to the limitations of power input, the power per unit volume decreases with the scale and as a result the mixing time increases with the fermentor volume. The longer mixing times in large fermentors cause DO concentration gradients. The importance of this variable was noted for penicillin and cephalosporin production. In both cases, there was a minimum threshold for DO concentration, below which the titers were strongly reduced.

188 Applied Microbiology: Industrial | Antibiotic Production

In the case of the former, it was 30% of saturation and that of the latter, 20%. A positive effect on the yield was seen if DO was maintained in the range 50–100%. As mentioned earlier, there is a limit to increase in power input and in superficial velocity of air. In such cases, either the back-pressure can be increased or the air stream can be enriched with oxygen. Both methods increase C and thereby enhance the driving force for OTR. It is a common practice to operate the fermentors with back-pressure. Just as the concentration of DO is critical to obtain high yields so is that of carbon dioxide. It is highly soluble in aqueous media and affects its pH. It can also inhibit production. Therefore, the aeration and agitation also have to be optimized/controlled to efficiently strip carbon dioxide from the medium. Another critical property of the fermentation broth that affects aeration is its rheology. Microorganisms belonging to different classes such as unicellular bacteria, actinomycetes, and molds have been used for the production of antibiotics. However, the majority of them are produced either by actinomycetes or by molds. Both these types of organisms grow in mycelial form, that is the ratio of length to diameter is very high. The fermentation broths containing mycelium are, even at low solids concentration, of non-Newtonian characteristics, that is their viscosity is not constant. The rheological properties of the fermentation broths significantly affect efficiency of aeration, agitation, and mixing. In viscous broth, significant gradients of DO, nutrients, pH, and temperature are observed. These often adversely affect the yield of the desired product. Furthermore, broth viscosity is affected by the culture morphology, which in turn changes with the age of the fermentation. Therefore, it is very important to study this parameter during the development of the fermentation process. A number of mathematical models have been proposed to explain the behavior of non-Newtonian fluids. These models have also been applied to mycelial broths. The usefulness of these models is that they allow prediction of rheological behavior of the broths in early stage of process development, which is important from an engineering point of view and assists in selection of strains more amenable to scale-up. In general, shorter mycelial length results in lower viscosity. Fungi can grow as pellets. However, as in the case of mycelial growth, that in the form of pellets also has its drawbacks. These are slower growth, lower reproducibility, and potential for DO limitation. It is also difficult to get good samples because the pellets settle quickly. In spite of these shortcomings, often the change over from mycelium to pellets is preferred since it results in lower viscosity and thus decreases the power required for good mixing and oxygen transfer. Pellet characteristics can be influenced by inoculum concentration and morphological characteristics, trace elements in the medium, polymer

additives to the medium and agitation (shear sensitivity of the culture). Sometimes it is not possible to reduce the broth viscosity by controlling the morphology of the culture. In such cases, the broth is diluted with water. This simple and practical technique reduces the viscosity significantly. It was found that a 10% dilution resulted in a 50% drop in viscosity.

Sterilization of Medium In the laboratory, the medium is sterilized by autoclaving, usually at 121  C, in a batch process. The time required to come up to the sterilization temperature and cool down to operating temperature is relatively short and does not significantly affect yield. However, at the higher scale these are significantly longer and lead to the destruction of temperature-sensitive medium constituents and increased levels of undesirable reactions between the medium components, such as those between reducing sugars and free amino groups. To avoid these problems, continuous sterilization, also called high temperature short time sterilization (HTST), is preferred over the batch type. As the name suggests the medium is rapidly heated to the desired high temperature (140  C or above) by pumping the medium through a heat exchanger or by steam injection. If the latter approach is used, it is important to correct for the dilution of the medium caused by the condensation of steam. The heated medium is held at the sterilization temperature for the desired time and cooled down quickly in a heat exchanger. To make the process economically efficient, the heat is recovered in the cooling stage by preheating the incoming medium. Medium sterilization was a critical variable for obtaining good yield of the antibiotic efrotomycin produced by N. lactamdurans. Glucose had to be sterilized with the rest of the medium ingredients. If sterilized separately, low or no efrotomycin was produced.

Heat Transfer Removal of heat generated by metabolic activity is a serious problem in large vessels. The present design of jacketed fermentors limits surface area available for transfer of heat from the medium to the cooling water. A number of other designs, such as hollow baffles, have been used to increase the surface area. In the geographic areas where cooling water temperatures in summer can be high, severe heat-transfer problems are encountered. Therefore, it is important to measure the heat load at the pilot plant level so adjustments can be made to the medium to reduce the peak rate of metabolism at the production scale.

Applied Microbiology: Industrial | Antibiotic Production

Process Control While processes to produce penicillin and streptomycin were being developed, the only sensors available were for monitoring temperature and pH. The next probe that was designed was a sterilizable DO probe. Analysis of exhaust gases became possible with the development of mass spectrometry for this purpose. These sensors and instruments provided information on the critical variables in a fermentation process. Later, with the availability of computers, it became possible to control the process based on the real-time values of the variables to maintain them in the optimum range. It also made it feasible to use different strategies such as cascade control to keep DO above the critical value. This was accomplished by controlling two variables, namely the rate of aeration and that of agitation. This is very useful in the case of a shear-sensitive producing culture. The rate of agitation can be increased up to a point above which shear will damage the culture. If needed, further increase in OTR can be obtained by increasing the airflow or increasing the back-pressure or both. The pH is controlled by the addition of acid or base. The location of the pH probe and the addition tube(s) is critical for successful control. The probe should be placed in the well-mixed region of the tank. Second, the acid or base should be added near one of the impellers, resulting in rapid dispersal. Addition of acid or base on the top surface of the medium, where bulk mixing is the worst in the tank, will result in their slow distribution and lead to local pH gradients with potentially deleterious effects on product formation. Sometimes ammonia gas mixed with incoming air is used to control pH. It is important to note that it can change the C/N ratio of the medium and may have a detrimental effect on the yield. Metabolism can be monitored in real time with exhaust gas analysis. The concentrations of carbon dioxide, oxygen, and nitrogen in the exhaust gas can be accurately measured and used to determine a number of critical parameters, such as carbon dioxide evolution rate, oxygen uptake rate, respiratory quotient, dry cell weight, and specific growth rate. In addition, this information can be utilized to initiate/control nutrient feeding, adjustment of batch temperature, and control aeration and agitation.

Downstream Processing The isolation/purification procedures consist of a combination of what is called unit operations in chemical engineering. These can be divided into four different groups: 1. removal of solids: filtration, centrifugation; 2. isolation of products: solvent extraction, adsorption;

189

3. purification: various types of chromatography, precipitation; 4. polishing or final purification: crystallization, drying. The choice of the unit operations to be used depends on the following: 1. characteristics of the antibiotic: solubility, ionic charge, stability and sensitivity to pH and/or temperature; 2. characteristics of the mycelium of the producer microorganism: whether it grows as a mycelial mat or pellets; 3. the location of the antibiotic: extracellular or cellassociated; 4. properties of the broth: presence of product-related impurities, medium components, proteins, polysaccharides; 5. purity of the starting material: normally low, between 0.1 and 1.0%; 6. quality of the final product required; 7. economics; 8. other factors such as available equipment, infrastructure, and so on. Based on these consideration, the operations are chosen and applied to develop a small-scale process, which is then scaled up to the pilot plant and finally to the production plant. Some examples are given in Table 3. It can be seen from the table that essentially the same unit operations mentioned above have been used to isolate and purify different antibiotics. However, the order in which they are used, the number of times each is used, the types of adsorbents and eluents used, the types of stationary phases or the solvents used in chromatography are all different for each of the antibiotics. Not only are these tailored for each of them, they vary among manufacturers. It is therefore really not possible to give a definitive downstream process for any one of the antibiotics, so rather an attempt is made to introduce the reader to the basic principles of it.

Conclusions The antibiotics have been our best weapon against deadly infectious diseases, which in the preantibiotic era caused large numbers of deaths. However, in the last 30 years, we have not been able to add to our arsenal any new class of compound, which acts on a novel target. In addition, this lack of success and economic considerations has led to reduction in effort to find new antibiotics. During the same period, pathogens have become resistant to the commonly used antibiotics, for example, methicillinresistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE). Thus, clearly there is a medical need for new antibiotics. On the brighter side, we now have better understanding of microbiology, biochemistry,

190 Applied Microbiology: Industrial | Antibiotic Production Table 3 Downstream processing of antibiotics Antibiotic

Class

Unit operations used

Penicillin

-Lactam

Cephalosporin Erythromycin

-Lactam Macrolide

Tetracyclines Streptomycin Rifamycin Lincomycin Chloramphenicol

Polycyclic structures Aminoglycoside Naphthalene-type Lincosamides p-Nitrophenol connected to dichloroacetamide

Filtration, solvent extraction, adsorption, elution, crystallization, drying Filtration, adsorption, chromatography, crystallization, drying Filtration, solvent extraction, adsorption, chromatography, crystallization, drying Filtration, adsorption, solvent extraction, crystallization, drying Filtration, adsorption, chromatography, precipitation, drying Filtration, solvent extraction, crystallization, drying Filtration, solvent extraction, crystallization, drying Filtration, solvent extraction, adsorption, crystallization, drying

and molecular biology and as a result we can design new approaches to finding and developing novel antibiotics. There was a report last year of a new antibiotic, platensimycin, which belonged to a new chemical class, had a new target, namely fatty acid biosynthesis and was developed using a new type of screen. The old approach of making new derivatives of older antibiotics is still productive and a number of such compounds are in clinical trials. While there is some debate whether bacterial genomics will result in new compounds, it has potential to help us to identify better targets and to identify and develop new antibiotics. A better future certainly awaits for the discovery of new anti-infective agents. See also: Aminoglycosides, Bioactive Bacterial Metabolites; Antibiotic Resistance; Macrolides; Polyketides; Quinolones; Strain Improvement; Streptomyces; -lactam antibiotics

Further Reading Baltz RH, McHenney MA, Cantwell CA, Queener SW, and Solenberg PJ (1997) Application of transposition mutagenesis in antibiotic producing streptomyces. Antoine Van Leeuwenhoek 71: 179–187. Berg DE and Berg CM (1983) The prokaryotic transposable element Tn5. Bio/Technology 1: 417–436. Bryskier A (2005) Historical review of antibacterial chemotherapy. In: Bryskier A (ed.) Antimicrobial Agents Antibacterial and Antifungals, 2nd edn., pp. 1–12. Washington, DC: ASM Press. Cooper CM, Fernstrom A, and Miller SA (1944) Performance of agitated gas–liquid contactors. Industrial and Engineering Chemistry 36: 504–509. Demain AL and Adrio JL (2008) Strain improvement for production of pharmaceuticals and other microbial metabolites. In: Petersen F and Amstutz R (eds.) Natural Compounds as Drugs, vol. I, pp. 251–289. Basel: Birkhauser Verlag AG.

DeVito JA, Mills JA, Liu VG, et al. (2002) An array of target-specific screening strains for antibacterial discovery. Nature Biotechnology 20: 478–483. Forsyth RA, Haselbeck RJ, Ohlsen KL, et al. (2002) A genome-wide strategy for the identification of essential genes in Staphylococcus aureus Molecular Microbiology 43: 1387–1400. Greasham RL and Herber WK (1997) Design and optimization of growth media. In: Rhodes PM and Stanbury PF (eds.) Applied Microbial Physiology, pp. 53–74. New York: IRL Press. Hunter JC, Fonda M, Sotos L, Toso B, and Belt A (1984) Ecological approaches to in isolation. Developments in Industrial Microbiology 25: 247–267. Hunter-Cevera JC and Belt A (1999) Isolation of cultures. In: Atlas RM, Cohen G, Hershberger RC, et al. (eds.) Manual of Industrial Microbiology and Biotechnology, 2nd edn., pp. 3–20. Washington, DC: ASM Press. Martin JF and Demain AL (1980) Control of antibiotic biosynthesis. Microbiological Reviews 44: 232–251. Masurekar PS (2005) Strain improvement for the production of secondary metabolites. In: An Z (ed.) Handbook of Industrial Mycology, pp. 539–561. New York: Marcel Dekker. Masurekar PS (2008) Nutritional and engineering aspects of microbial process development. In: Petersen F and Amstutz R (eds.) Natural Compounds as Drugs, vol. I, pp. 291–328. Basel: Birkhauser Verlag AG. Monaghan RL and Barrett JF (2006) Antibacterial drug discovery-then, now and the genomics future. Biochemical Pharmacology 71: 901–909. Myers RH and Montgomery DC (2002) Response Surface Methodology. New York: John Wiley and Sons. Vandamme EJ (ed.) (1984) Biotechnology of Industrial Antibiotics. New York: Marcel Dekker. Wu CFJ and Hamada M (2000) Experiments Planning, Analysis and Parameter Design Optimization. New York: John Wiley and Sons.

Relevant Website http://www.ironwoodpharma.com – Microbia is now Ironwood pharmaceuticals.