- Email: [email protected]
Microbial biotechnology
FEATURES 10 Hoogendoorn, B. et al. (1999) Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography. Hum. Genet. 104, 89–93 11 Lander, E.S. (1999) Array of hope. Nat. Genet. 21, 3–4 12 Syvanen, A-C. et al. (1993) Identification of individuals by analysis of bi-allelic DNA markers, using PCR and solid-phase minisequencing.
Am. J. Hum. Genet. 52, 46–59 13 Griffin, T.J. et al. (1999) Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 96, 6301–6306 14 Gibbs, R.A. et al. (1989) Detection of single DNA base differences by competitive oligonucleotide priming. Nucleic Acids Res. 17, 2437–2448
Microbial biotechnology Arnold L. Demain For thousands of years, microorganisms have been used to supply products such as bread, beer and wine. A second phase of traditional microbial biotechnology began during World War I and resulted in the development of the acetone-butanol and glycerol fermentations, followed by processes yielding, for example, citric acid, vitamins and antibiotics. In the early 1970s, traditional industrial microbiology was merged with molecular biology to yield more than 40 biopharmaceutical products, such as erythropoietin, human growth hormone and interferons. Today, microbiology is a major participant in global industry, especially in the pharmaceutical, food and chemical industries.
icroorganisms are important for many reasons, particularly because they produce things that are of value to us1. These can be very large materials (e.g. proteins, nucleic acids, carbohydrate polymers, even cells) or smaller molecules and are usually divided into metabolites that are essential for vegetative growth (primary) and those that are inessential (secondary). Although microbes are extremely good at producing an amazing array of valuable products, they usually produce these compounds in small amounts that are needed for their own benefit. Regulatory mechanisms have evolved that enable a strain to avoid excessive production of its metabolites so that it can compete efficiently with other forms of life and survive in nature. By contrast, the industrial microbiologist screens for a ‘wasteful’ strain that will overproduce a particular compound that can be isolated and marketed. After a desired strain has been found, a development program is initiated to improve titers by modification of culture conditions using mutation and recombinant DNA techniques. The main reason for the use of microorganisms to produce compounds that can otherwise be isolated from plants and animals, or synthesized by chemists, is the ease of increasing production by environmental and genetic manipulation; 1000-fold increases have been recorded for small metabolites2.
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Traditional microbial biotechnology Primary metabolites Primary metabolites are the small molecules of living cells; they are intermediates or end products of the pathways of intermediary metabolism, building blocks for essential macromolecules, or are converted into A.L. Demain ([email protected]) is at the Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
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coenzymes. Primary metabolites used in the food and feed industries include: alcohols (ethanol), amino acids (monosodium glutamate, lysine, threonine, phenylalanine, tryptophan), flavor nucleotides (59-guanylic acid, 59-inosinic acid), organic acids (acetic, propionic, succinic, fumaric, lactic), polyols (glycerol, mannitol, erythritol, xylitol), polysaccharides (xanthan, gellan), sugars (fructose, ribose, sorbose) and vitamins [riboflavin (B2), cyanocobalamin (B12), biotin]. Mutants During amino acid production, feedback regulation is bypassed by isolating auxotrophic mutants and partially starving them of their requirements. Another method is to produce mutants that are resistant to a toxic analog of the desired metabolite, that is, an antimetabolite. Combinations of auxotrophic and antimetabolite resistance mutations are common in primary metaboliteproducing microorganisms. Fermentation Another factor is the increase in outward permeability, which is very important in the production of L-glutamic acid, the major commercial amino acid. Approximately 1.2 billion pounds of monosodium glutamate are made annually by fermentation using various species of the genera Corynebacterium (e.g. C. glutamicum) and Brevibacterium (e.g. B. flavum and B. lactofermentum). Molar yields of glutamate from sugar are 50–60% and broth concentrations reach over 100 g l21. Glutamic acid Normally, glutamic acid overproduction would not occur because of feedback regulation. However, modification of the cell membrane can cause glutamate to be pumped out of the cell, thus allowing its biosynthesis
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to proceed unabated. This membrane alteration is intentionally effected by biotin limitation (all glutamic acid bacteria are biotin auxotrophs), glycerol limitation of glycerol auxotrophs, oleate limitation of oleate auxotrophs, or addition of penicillin or fatty acid derivatives to exponentially growing cells. Apparently, all of these manipulations result in a phospholipiddeficient cytoplasmic membrane. The excretion is carried out by a specific efflux system involving a carrier that is dependent on membrane potential. Lysine Most cereals are deficient in the essential amino acid L-lysine. Lysine is a member of the aspartate family of amino acids and is produced in bacteria by a branched pathway that also produces methionine, threonine and isoleucine. This pathway is controlled very tightly in an organism such as Escherichia coli, which includes three aspartate kinases that are each regulated by a different end product. In addition, after each branch point, the initial enzymes are inhibited by their respective end products and no overproduction occurs. However, in lysine-fermentation organisms (e.g. various mutants of C. glutamicum and its relatives), there is only a single aspartate kinase, which is regulated via concerted feedback inhibition by threonine and lysine. By the genetic removal of homoserine dehydrogenase, a glutamate-producing wild-type Corynebacterium is converted into a lysine-overproducing mutant that cannot grow unless methionine and threonine are added to the medium. As long as the threonine supplement is maintained at a limiting concentration, the intracellular concentration of threonine is the limiting factor and feedback inhibition of aspartate kinase is bypassed. In addition to the difference in the mode of aspartate kinase feedback inhibition, lysine overproducers differ from E. coli in the following ways: (1) no feedback repression of aspartate kinase or aspartate semialdehyde dehydrogenase occurs in lysine overproducers; (2) the first and second enzymes of the lysine branch (dihydrodipicolinate synthetase and dihydrodipicolinate reductase) are neither inhibited nor repressed by lysine in lysine overproducers; and (3) L-lysine decarboxylase is absent in lysine overproducers. Lysine excretion is via active transport involving a carrier and is driven by membrane potential, the lysine gradient and the proton gradient. Excretion uses a (2OH2)–lysine symporter and is catalysed by a dipeptide-uptake system dependent on electromotive force, not on ATP. World markets for amino acids amount to US$915 million for L-glutamate, US$450 million for L-lysine, US$198 million for L-phenylalanine and US$43 million for L-aspartate. Recombinant DNA technology Recombinant DNA technology is beginning to have a major impact on amino acid production3,4. The major manipulation for lysine production is aimed at increasing the levels of feedback-resistant aspartate kinase and dihydrodipicolinate synthase. As a result, lysine industrial production yields 120 g l21 and 0.25–0.35 g L-lysine • HCl g21 glucose used (molar yield of 0.25–0.35 mol of L-lysine mol21 of glucose used). Recombinant technology and traditional mutagenesis, plus selection, have been major influences in constructing bacterial strains capable of producing these levels (in g l21) TIBTECH JANUARY 2000 (VOL 18)
of amino acids: L-threonine, 100; L-isoleucine, 40; L-leucine, 34; L-valine, 31; L-phenylalanine, 28; L-tryptophan, 55; L-tyrosine, 26; L-proline, 100; L-arginine, 100; and L-histidine, 40. Commercial interest in nucleotide fermentations is due to the activity of two purine ribonucleoside 59-monophosphates, namely guanylic acid (GMP) and inosinic acid (IMP), as flavor enhancers. Approximately 2500 tons of GMP and IMP are produced annually in Japan alone, with a world market of US$350 million. Techniques similar to those described above for amino acid fermentations have yielded IMP titers of 27 g l21. Vitamins Riboflavin (vitamin B2) overproducers include two yeast-like molds, Eremothecium ashbyii and Ashbya gossypii, which synthesize riboflavin in concentrations greater than 20 g l21. New processes using Candida sp. or recombinant Bacillus subtilis strains that produce up to 30 g l21 riboflavin have been developed in recent years. Vitamin B12 is produced on an industrial scale by Propionibacterium shermanii or Pseudomonas denitrificans. The key to the fermentation is to avoid feedback repression by vitamin B12. The early stage of the P. shermanii fermentation is conducted under anaerobic conditions in the absence of the precursor 5,6-dimethylbenzimidazole. These conditions prevent vitamin B12 synthesis and allow for the accumulation of the intermediate, cobinamide. The culture is then aerated and dimethylbenzimidazole is added, converting cobinamide to the vitamin. In the P. denitrificans fermentation, the entire process is carried out under low oxygen. A high level of oxygen results in an oxidizing intracellular environment that represses the formation of the early enzymes in the pathway. Production of vitamin B12 has reached levels of 150 mg l21 and a world market value of US$71 million. During production of biotin, feedback repression is caused by the enzyme protein acetyl-coenzyme A carboxylase biotin holoenzyme synthetase, with biotin 5-adenylate acting as corepressor. Strains of Serratia marcescens obtained by mutagenesis, selection for resistance to biotin antimetabolites, and molecular cloning can produce 600 mg l21 in the presence of high concentrations of sulfur and ferrous iron. Such a titer is high enough to economically compete with the traditional chemical production process. Fungi Filamentous fungi are widely used for the commercial production of organic acids, for example, 1 billion pounds of citric acid are produced per year with a market value of US$1.4 billion. Citric acid is produced via the Embden-Meyerhof pathway and the first step of the tricarboxylic acid cycle; the control of the process involves the inhibition of phosphofructokinase by citric acid. The commercial process uses Aspergillus niger in media deficient in iron and manganese. A high level of citric acid production is also associated with a high intracellular concentration of fructose 2,6-biphosphate, an activator of glycolysis. Other factors contributing to high citric acid production are the inhibition of isocitrate dehydrogenase by citric acid and the low optimum pH (1.7–2.0). Higher pH values (e.g. 3.0) lead to the production of
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oxalic and gluconic acids, instead of citric acid. The low pH inactivates glucose oxidase, which would normally yield gluconic acid. In approximately 4–5 days, the major proportion (80%) of the sugar is converted to citric acid, with titers reaching about 100 g l21. Alternative processes have been developed for the production of citric acid by Candida yeasts, especially from hydrocarbons. Such yeasts are able to convert n-paraffins to citric and isocitric acids in extremely high yields [150–170% (w/w) of substrate used]; titers as high as 225 g l21 have been reached. Alcohol Ethyl alcohol is a primary metabolite produced by fermentation of sugar, or a polysaccharide that can be depolymerized to a fermentable sugar. Saccharomyces cerevisiae is used for the fermentation of hexoses, whereas Kluyveromyces fragilis or Candida species can be used if lactose or a pentose, respectively, is the substrate. Under optimum conditions, approximately 10–12% ethanol by volume is obtained within five days. Such a high concentration slows down growth and the fermentation ceases. With special yeasts, the fermentation can be continued to produce alcohol concentrations of 20% by volume, but these concentrations are attained only after months or years of fermentation. At present, all beverage alcohol is made by fermentation. Industrial ethanol is mainly manufactured by fermentation, but some is produced from ethylene by the petrochemical industry. Bacteria such as clostridia and Zymomonas are being re-examined for ethanol production after years of neglect. Clostridium thermocellum, an anaerobic thermophile, can convert waste cellulose (i.e. biomass) and crystalline cellulose directly to ethanol. Other clostridia produce acetate, lactate, acetone and butanol, and will be used to produce these chemicals when the gobal petroleum supplies begin to become depleted. Fuel ethanol produced from biomass would provide relief from air pollution caused by the use of gasoline and would not contribute to the greenhouse effect. E. coli has been converted into an excellent ethanol producer (43% yield, v/v) by recombinant DNA techniques. Bioconverting-organisms In addition to the multireaction sequences of fermentations, microorganisms are extremely useful in carrying out biotransformation processes in which a compound is converted into a structurally related product by one or a small number of enzymes contained in the cells5. Bioconverting-organisms are known for practically every type of chemical reaction. The reactions are stereospecific; the ultimate in specificity is exemplified by steroid bioconversions. Bioconversions are characterized by extremely high yields, approximately 90–100%. Other attributes include mild reaction conditions and the coupling of reactions, using a microorganism containing several enzymes working in series. Secondary metabolites Microbially produced secondary metabolites6 are extremely important for health and nutrition. As a group that includes antibiotics, other medicinals, toxins, biopesticides and animal and plant growth factors, they
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have tremendous economic importance. Secondary metabolites have no function in the growth of the producing cultures (although, in nature, they are essential for the survival of the producing organism), are produced by certain restricted taxonomic groups of organisms and are usually formed as mixtures of closely related members of a chemical family. Antibiotics The best-known group of the secondary metabolites are the antibiotics7. Their targets include DNA replication (actinomycin, bleomycin and griseofulvin), transcription (rifamycin), translation by 70-S ribosomes (chloramphenicol, tetracycline, lincomycin, erythromycin and streptomycin), transcription by 80-S ribosomes (cyclohexamide), transcription by 70- and 80-S ribosomes (puromycin and fusidic acid), cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin and vancomycin) and cell membranes (surfactants including: polymyxin and amphotericin; channelforming ionophores, such as linear gramicidin; and mobile carrier ionophores, such as monensin). Since 1940, there has been a virtual explosion of new and potent antibiotic molecules that have been of great use in medicine, agriculture and basic research. In 1996, the antibiotic market was composed of 160 antibiotics and amounted to a world market value of ~US$23 billion. The search for new antibiotics continues, in order to: combat evolving pathogens, naturally resistant bacteria and fungi, and previously susceptible microbes that have developed resistance; improve pharmacological properties; combat tumors, viruses and parasites; and discover safer, more potent and broaderspectrum compounds. In the search for new antibiotics, many of the new products are made chemically by modification of natural antibiotics via semisynthesis. Antibiotics are used not only for chemotherapy in human and veterinary medicine, but also for growth promotion in farm animals and for the protection of plants. Non-antibiotic agents In nature, secondary metabolites are important to the organisms that produce them, functioning as: (1) sex hormones; (2) ionophores; (3) competitive weapons against other bacteria, fungi, amoebae, insects and plants; (4) agents of symbiosis; and (5) effectors of differentiation. For years, most pharmaceuticals that were used for non-infectious diseases were strictly synthetic products8. Similarly, most therapeutics for nonmicrobial parasitic diseases in animals (e.g. coccidiostats and antihelminthics) came from the screening of synthesized compounds followed by molecular modification. Despite the testing of thousands of synthetic compounds, only a few promising structures were found. As new lead compounds became more and more difficult to find, microbial broths filled the void and microbial products increased in importance in the therapy of non-microbial diseases9. Today, microbially produced polyethers such as monensin, lasalocid and salinomycin dominate the coccidiostat market and are also the chief growth promoters in use for ruminant animals. The avermectins, another group of streptomycete products with a market of more than US$1 billion per year, have high activity against helminths and arthropods. TIBTECH JANUARY 2000 (Vol. 18)
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Many microbial products with important pharmacological activities were discovered by screening for inhibitors using simple enzymatic assays. One huge success has been the statins, including lovastatin (also known as mevinolin) and pravastatin: fungal products that are used as cholesterol-lowering agents in humans and animals. In its hydroxy acid form, lovastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from liver. Other wellknown enzyme inhibitors include: clavulanic acid, a penicillinase-inhibitor that protects penicillin from inactivation by resistant pathogens; and acarbose, a natural inhibitor of intestinal glucosidase, which is produced by an actinomycete of the genus Actinoplanes. Acarbose decreases hyperglycemia and triglyceride synthesis in adipose tissue, the liver and the intestinal wall of patients suffering from diabetes, obesity and type IV hyperlipidemia. Biopesticides Also in commercial or near-commercial use are biopesticides, including biofungicides (e.g. kasugamycin, polyoxins), bioinsecticides (nikkomycin, spinosyns), bioherbicides (bialaphos), antihelminthics (avermectin), coccidiostats, ruminant-growth promoters (monensin, lasalocid, salinomycin), plant-growth regulators (gibberellins), immunosuppressants for organ transplants (cyclosporin A, FK-506, rapamycin), anabolic agents in farm animals (zearelanone), uterocontractants (ergot alkaloids) and antitumor agents (doxorubicin, daunorubicin, mitomycin, bleomycin). Tropophase and idiophase In batch culture, most secondary metabolite processes have a distinct growth phase (trophophase) followed by a production phase (idiophase). In other fermentations, the two phases overlap; the timing depends on the nutritional environment presented to the culture, the growth rate, or both. A delay in antibiotic production until after trophophase helps the producing organism because the microbe is sometimes sensitive to its own antibiotic during growth. Resistance mechanisms that develop in producing microorganisms include enzymatic modification of the antibiotic, alteration of the cellular target of the antibiotic and decreased uptake of the excreted antibiotic. Directed biosynthesis The manipulation of the culture media in any development program often involves the testing of hundreds of additives as possible limiting precursors of the desired product. Occasionally, a precursor that increases production of the secondary metabolite is found. The precursor may also direct the fermentation towards the formation of one specific desirable product: this is known as directed biosynthesis. Examples of directed biosynthesis include the use of phenylacetic acid in the fermentation of benzylpenicillin, and specific amino acids in the production of actinomycins and tyrocidins. Stimulatory precursors include: methionine, as an inducer in cephalosporin C formation; valine, in tylosin production; and tryptophan for ergot-alkaloid production. In many fermentations, however, precursors show no activity because their syntheses are not rate-limiting. In such cases, TIBTECH JANUARY 2000 (Vol. 18)
screening of additives has often revealed dramatic effects, both stimulatory and inhibitory, of non-precursor molecules on the production of secondary metabolites. These effects are usually due to the interaction of these compounds with the regulatory mechanisms existing in the fermentation organism. Antibiotic biosynthesis ends via the decay of antibiotic synthetases or because of feedback inhibition and repression of these enzymes. Because the regulatory mechanisms are genetically determined, mutations have had a major effect on the production of secondary metabolites. Indeed, it is the chief factor responsible for the 100–1000-fold increases obtained in the production of antibiotics from their initial discovery to the present time. These tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by random mutagenesis and screening for higher-producing microbial strains. Mutation has also served to: (1) shift the proportion of metabolites produced in a fermentation broth to a more favorable distribution; (2) elucidate the pathways of secondary metabolism; and (3) yield new compounds. Modern microbial biotechnology Modern biotechnology is now over 25 years old10. In 1972, the birth of recombinant DNA technology propelled biotechnology to new heights and led to the establishment of a new industry. In addition to recombinant DNA technology, modern microbial biotechnology encompasses fermentation, microbial physiology, high-throughput screening for novel metabolites and strain improvement, bioreactor design and downstream processing, cell immobilization (enzyme engineering), cell fusion, metabolic engineering, bioreactor design, downstream processing, in vitro mutagenesis (protein engineering) and directed evolution of enzymes (applied molecular evolution). Recombinant microorganisms The revolutionary exploitation of microbial genetic discoveries in the 1970s, 1980s and 1990s depended heavily upon the solid structure of industrial microbiology, described above. The major microbial hosts for production of recombinant proteins are E. coli11, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorpha and Aspergillus niger. The use of recombinant microorganisms (Fig. 1) provided the techniques and experience necessary for the successful application of higher organisms, such as mammalian and insect cell culture, and transgenic animals and plants as hosts for the production of glycosylated recombinant proteins. Progress The progress in biotechnology has been truly remarkable. Within four years of the discovery of recombinant DNA technology, genetically engineered bacteria were making human insulin and human growth hormone. This led to an explosion of investment activity in new companies, mainly dedicated to innovation via genetic approaches. Newer companies entered the scene in various niches such as biochemical engineering and downstream processing. Today, biotechnology in the USA is represented by some 1300 companies with revenues of US$19.6 billion, of which sales represent US$13.4 billion, and there are
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was that of hepatitis B virus surface antigen produced in yeast. The great contribution made by recombinant vaccines is the elimination of the tragic problems associated with conventional vaccines. Through reversion of the attenuated pathogen, some individuals receiving the conventional vaccine not only failed to be protected, but also came down with the disease. Combinatorial biosynthesis As mentioned previously, recombinant DNA techniques have made a significant impact on the production of vitamins, amino acids, nucleotides, bioconversion and secondary metabolites. Most microbial biosynthetic pathways are encoded by clustered genes, which facilitates the transfer of an entire pathway in a single manipulation. Even in fungi, pathway genes are sometimes clustered, such as the penicillin genes in Penicillium or the aflatoxin genes in Aspergillus. For the discovery of new or modified secondary products, recombinant DNA techniques are being used to introduce genes for the synthesis of one product into producers of other antibiotics or into non-producing strains (combinatorial biosynthesis).
Figure 1 Escherichia coli: the workhorse of modern microbial biotechnology. (Electron micrograph taken by Erika Hartweig, bar = 1 mm.)
approximately 153 000 employees. The number of biotechnology companies in Canada reached 282 in 1998, employing 10 000 workers and with revenues of approximately US$1.1 billion. Japan’s biotechnology sales were approximately US$10 billion, mainly by established pharmaceutical, food and beverage companies. European biotechnology moved rapidly in the 1990s, after years of lagging behind and, in 1998, 1178 biotechnology companies existed with 45 000 employees, and revenues of US$3.7 billion. The major thrust of recombinant DNA technology has been in the area of rare mammalian peptides, such as hormones, growth factors, enzymes, antibodies and biological response modifiers12. Among those genetically engineered products that have been approved for use in the USA are human insulin, human growth hormone, erythropoietin, antihemophelia factor, granulocyte-colony stimulating factor, granulocytemacrophage-colony stimulating factor, epidermal growth factor and other growth factors, interleukin-2, a-, b- and g-interferons, and bovine somatotropin. Vaccines Vaccine production is another important part of the new technology; the first subunit vaccine on the market
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Enzyme production The production of enzymes by fermentation was an established business before modern microbial biotechnology. However, recombinant DNA methodology was so perfectly suited to the improvement of enzymeproduction technology that it was almost immediately used by companies involved in manufacturing enzymes. Industrial enzymes have now reached an annual market of US$1.6 billion. Important enzymes are proteases, lipases, carbohydrases, recombinant chymosin for cheese manufacture and recombinant lipase for use in detergents. Recombinant therapeutic enzymes already have a market value of over US$2 billion, being used for thromboses, gastrointestinal and rheumatic disorders, metabolic diseases and cancer. They include tissue plasminogen activator, human DNAase and Cerozyme. Agriculture Industrial microbiology through genetic engineering and its associated disciplines has brought about a revolution in agriculture. Two bacteria have had a major influence: Agrobacterium tumefaciens, a bacterium that normally produces crown gall tumors on dicotyledonous plants; and Bacillus thuringiensis, an insecticidal bacterium. The tumor-forming genes of A. tumefaciens are present on its tumor-inducing (Ti) plasmid, along with genes directing the plant to form opines (nutritional factors required by the bacterium that it cannot produce by itself ). The Ti vector has been exceedingly valuable for introducing foreign genes into dicotyledonous plants for production of transgenic plants. However, the Ti plasmid is not very successful for transferring genes into monocotyledonous plants, a problem bypassed by, for example, the development of a particleacceleration gun, which shoots DNA-coated metal particles into plant cells. The activity of the insecticidal bacterium, B. thuringiensis, is caused by its crystal protein produced during sporulation. Crystals and spores have been applied to plants for many years to protect them against lepidopteran insects. B. thuringiensis preparations are highly potent, approximately 300 times more active TIBTECH JANUARY 2000 (Vol. 18)
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on a molar basis than synthetic pyrethroids and 80 000 times more active than organophosphate insecticides. In the modern biotechnology era, plants resistant to insects have been produced by expressing forms of the B. thuringiensis toxin gene in the plant. Recently developed bioinsecticides include insect viruses, such as baculoviruses, that are engineered to produce arthropod toxins. Transgenic plants, resistant to herbicides, are also available, as are virus-resistant plants produced by expressing viral-coat-protein genes in plants. Interestingly, chemical pesticides against plant viruses were never available. Conclusion Although most of the early promises of biotechnology have been achieved, major challenges remain. We must use our brains, technology, drive and dedication to solve the problems of evolving diseases (e.g. AIDS), established diseases (cancer and parasitic infection), antibioticresistance development and environmental pollution, by converting urban, industrial and agricultural wastes into resources such as liquid fuel. These efforts will require continued interaction between different disciplines, major support by governments and international agencies, as well as an understanding and supportive public. References 1 Demain, A.L. (1990) Achievements in microbial technology. Biotechnol. Adv. 8, 291–301
2 Demain, A.L. (1988) Contributions of genetics to the production and discovery of microbial pharmaceuticals. Pure Appl. Chem. 60, 833–836 3 Jetten, M.S. and Sinskey, A.J. (1995) Recent advances in the physiology and genetics of amino acid-producing bacteria. Crit. Rev. Biotechnol. 15, 73–103 4 Sahm, H. et al. (1995) Metabolic design in amino acid producing bacterium Corynebacterium glutamicum. FEMS Microbiol. Rev. 16, 243–252 5 Kieslich, K. (1997) Biotransformations. In Fungal Biotechnology (Anke, T., ed.), pp. 297–399, Chapman & Hall 6 Demain, A.L. (1992) Microbial secondary metabolism: a new theoretical frontier for academia, a new opportunity for industry. In Secondary Metabolites: Their Function and Evolution (Chadwick, D.J. and Whelan, J., eds), pp. 3–23, John Wiley & Sons 7 Strohl, W.R. (1997) Biotechnology of Antibiotics, 2nd edn, Marcel Dekker 8 Demain, A.L. (1996) Fungal secondary metabolism: regulation and functions. In A Century of Mycology (Sutton, B., ed.), pp. 233–254, Cambridge University Press 9 Demain, A.L. (1998) Microbial natural products: alive and well in 1998. Nat. Biotechnol. 16, 3–4 10 Cohen, S.N. (1979) The transplantation and manipulation of genes in microorganisms. The Harvey Lectures 74, 173–204 11 Swartz, J.R. (1996) Escherichia coli recombinant DNA technology. In Escherichia coli and Salmonella: Cellular and Molecular Biology 2nd edn (Neidhardt, F.C., ed.), pp. 1693–1711, American Society of Microbiology (ASM) Press 12 Demain, A.L. et al. (1994) Contributions of recombinant microbes and their potential. In Recombinant Microbes for Industrial and Agricultural Applications (Murooka, T. and Imanaka, T., eds), pp. 27–46, Marcel Dekker
Drug discovery in the new millennium: the pivotal role of biotechnology Graham J. Boulnois Biotechnology has made, and will continue to make, a major contribution to the health of mankind by providing new insights into disease and acting as a pivotal enabler for the drug-discovery process. The available techniques are diverse and changing rapidly: selecting and integrating the best approach is the key to success.
odern medicines have improved the life of humankind, and the past few decades have seen enormous advances in our ability to treat, manage and prevent a large number of diseases. Successful therapies range from the treatment of acute infections with powerful antibiotics and the availability of anaesthetics for surgery and management, through risk reduction in cardiovascular disease to the prevention of a range of infectious diseases via vaccination. Biotechnology has already played a major role in modern medicine discovery, delivering a range of new treatments and vaccines in its own right, and gene therapy is just round the corner. Despite these impressive
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G.J. Boulnois ([email protected]) is at AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK SK10 4TG. TIBTECH JANUARY 2000 (Vol. 18)
advances, there are many diseases for which treatments are lacking or are imperfect, and we are a long way from eradicating or even reducing the prevalence of many debilitating conditions. In short, the opportunities for new treatments is huge. We have also seen a change in the way new medicines are discovered, driven by biotechnology in general but particularly by molecular biology. The reliance on testing new synthetic organic molecules in animals or in whole-organ preparations, as an early part of the discovery process, has changed to a molecular target approach in which in vitro screening of compounds against purified, recombinant proteins or genetically modified cell lines is carried out with a high throughput. This change has come about as a consequence of better and ever-improving knowledge of the molecular basis
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Am. J. Hum. Genet. 52, 46–59 13 Griffin, T.J. et al. (1999) Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 96, 6301–6306 14 Gibbs, R.A. et al. (1989) Detection of single DNA base differences by competitive oligonucleotide priming. Nucleic Acids Res. 17, 2437–2448
Microbial biotechnology Arnold L. Demain For thousands of years, microorganisms have been used to supply products such as bread, beer and wine. A second phase of traditional microbial biotechnology began during World War I and resulted in the development of the acetone-butanol and glycerol fermentations, followed by processes yielding, for example, citric acid, vitamins and antibiotics. In the early 1970s, traditional industrial microbiology was merged with molecular biology to yield more than 40 biopharmaceutical products, such as erythropoietin, human growth hormone and interferons. Today, microbiology is a major participant in global industry, especially in the pharmaceutical, food and chemical industries.
icroorganisms are important for many reasons, particularly because they produce things that are of value to us1. These can be very large materials (e.g. proteins, nucleic acids, carbohydrate polymers, even cells) or smaller molecules and are usually divided into metabolites that are essential for vegetative growth (primary) and those that are inessential (secondary). Although microbes are extremely good at producing an amazing array of valuable products, they usually produce these compounds in small amounts that are needed for their own benefit. Regulatory mechanisms have evolved that enable a strain to avoid excessive production of its metabolites so that it can compete efficiently with other forms of life and survive in nature. By contrast, the industrial microbiologist screens for a ‘wasteful’ strain that will overproduce a particular compound that can be isolated and marketed. After a desired strain has been found, a development program is initiated to improve titers by modification of culture conditions using mutation and recombinant DNA techniques. The main reason for the use of microorganisms to produce compounds that can otherwise be isolated from plants and animals, or synthesized by chemists, is the ease of increasing production by environmental and genetic manipulation; 1000-fold increases have been recorded for small metabolites2.
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Traditional microbial biotechnology Primary metabolites Primary metabolites are the small molecules of living cells; they are intermediates or end products of the pathways of intermediary metabolism, building blocks for essential macromolecules, or are converted into A.L. Demain ([email protected]) is at the Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
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coenzymes. Primary metabolites used in the food and feed industries include: alcohols (ethanol), amino acids (monosodium glutamate, lysine, threonine, phenylalanine, tryptophan), flavor nucleotides (59-guanylic acid, 59-inosinic acid), organic acids (acetic, propionic, succinic, fumaric, lactic), polyols (glycerol, mannitol, erythritol, xylitol), polysaccharides (xanthan, gellan), sugars (fructose, ribose, sorbose) and vitamins [riboflavin (B2), cyanocobalamin (B12), biotin]. Mutants During amino acid production, feedback regulation is bypassed by isolating auxotrophic mutants and partially starving them of their requirements. Another method is to produce mutants that are resistant to a toxic analog of the desired metabolite, that is, an antimetabolite. Combinations of auxotrophic and antimetabolite resistance mutations are common in primary metaboliteproducing microorganisms. Fermentation Another factor is the increase in outward permeability, which is very important in the production of L-glutamic acid, the major commercial amino acid. Approximately 1.2 billion pounds of monosodium glutamate are made annually by fermentation using various species of the genera Corynebacterium (e.g. C. glutamicum) and Brevibacterium (e.g. B. flavum and B. lactofermentum). Molar yields of glutamate from sugar are 50–60% and broth concentrations reach over 100 g l21. Glutamic acid Normally, glutamic acid overproduction would not occur because of feedback regulation. However, modification of the cell membrane can cause glutamate to be pumped out of the cell, thus allowing its biosynthesis
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to proceed unabated. This membrane alteration is intentionally effected by biotin limitation (all glutamic acid bacteria are biotin auxotrophs), glycerol limitation of glycerol auxotrophs, oleate limitation of oleate auxotrophs, or addition of penicillin or fatty acid derivatives to exponentially growing cells. Apparently, all of these manipulations result in a phospholipiddeficient cytoplasmic membrane. The excretion is carried out by a specific efflux system involving a carrier that is dependent on membrane potential. Lysine Most cereals are deficient in the essential amino acid L-lysine. Lysine is a member of the aspartate family of amino acids and is produced in bacteria by a branched pathway that also produces methionine, threonine and isoleucine. This pathway is controlled very tightly in an organism such as Escherichia coli, which includes three aspartate kinases that are each regulated by a different end product. In addition, after each branch point, the initial enzymes are inhibited by their respective end products and no overproduction occurs. However, in lysine-fermentation organisms (e.g. various mutants of C. glutamicum and its relatives), there is only a single aspartate kinase, which is regulated via concerted feedback inhibition by threonine and lysine. By the genetic removal of homoserine dehydrogenase, a glutamate-producing wild-type Corynebacterium is converted into a lysine-overproducing mutant that cannot grow unless methionine and threonine are added to the medium. As long as the threonine supplement is maintained at a limiting concentration, the intracellular concentration of threonine is the limiting factor and feedback inhibition of aspartate kinase is bypassed. In addition to the difference in the mode of aspartate kinase feedback inhibition, lysine overproducers differ from E. coli in the following ways: (1) no feedback repression of aspartate kinase or aspartate semialdehyde dehydrogenase occurs in lysine overproducers; (2) the first and second enzymes of the lysine branch (dihydrodipicolinate synthetase and dihydrodipicolinate reductase) are neither inhibited nor repressed by lysine in lysine overproducers; and (3) L-lysine decarboxylase is absent in lysine overproducers. Lysine excretion is via active transport involving a carrier and is driven by membrane potential, the lysine gradient and the proton gradient. Excretion uses a (2OH2)–lysine symporter and is catalysed by a dipeptide-uptake system dependent on electromotive force, not on ATP. World markets for amino acids amount to US$915 million for L-glutamate, US$450 million for L-lysine, US$198 million for L-phenylalanine and US$43 million for L-aspartate. Recombinant DNA technology Recombinant DNA technology is beginning to have a major impact on amino acid production3,4. The major manipulation for lysine production is aimed at increasing the levels of feedback-resistant aspartate kinase and dihydrodipicolinate synthase. As a result, lysine industrial production yields 120 g l21 and 0.25–0.35 g L-lysine • HCl g21 glucose used (molar yield of 0.25–0.35 mol of L-lysine mol21 of glucose used). Recombinant technology and traditional mutagenesis, plus selection, have been major influences in constructing bacterial strains capable of producing these levels (in g l21) TIBTECH JANUARY 2000 (VOL 18)
of amino acids: L-threonine, 100; L-isoleucine, 40; L-leucine, 34; L-valine, 31; L-phenylalanine, 28; L-tryptophan, 55; L-tyrosine, 26; L-proline, 100; L-arginine, 100; and L-histidine, 40. Commercial interest in nucleotide fermentations is due to the activity of two purine ribonucleoside 59-monophosphates, namely guanylic acid (GMP) and inosinic acid (IMP), as flavor enhancers. Approximately 2500 tons of GMP and IMP are produced annually in Japan alone, with a world market of US$350 million. Techniques similar to those described above for amino acid fermentations have yielded IMP titers of 27 g l21. Vitamins Riboflavin (vitamin B2) overproducers include two yeast-like molds, Eremothecium ashbyii and Ashbya gossypii, which synthesize riboflavin in concentrations greater than 20 g l21. New processes using Candida sp. or recombinant Bacillus subtilis strains that produce up to 30 g l21 riboflavin have been developed in recent years. Vitamin B12 is produced on an industrial scale by Propionibacterium shermanii or Pseudomonas denitrificans. The key to the fermentation is to avoid feedback repression by vitamin B12. The early stage of the P. shermanii fermentation is conducted under anaerobic conditions in the absence of the precursor 5,6-dimethylbenzimidazole. These conditions prevent vitamin B12 synthesis and allow for the accumulation of the intermediate, cobinamide. The culture is then aerated and dimethylbenzimidazole is added, converting cobinamide to the vitamin. In the P. denitrificans fermentation, the entire process is carried out under low oxygen. A high level of oxygen results in an oxidizing intracellular environment that represses the formation of the early enzymes in the pathway. Production of vitamin B12 has reached levels of 150 mg l21 and a world market value of US$71 million. During production of biotin, feedback repression is caused by the enzyme protein acetyl-coenzyme A carboxylase biotin holoenzyme synthetase, with biotin 5-adenylate acting as corepressor. Strains of Serratia marcescens obtained by mutagenesis, selection for resistance to biotin antimetabolites, and molecular cloning can produce 600 mg l21 in the presence of high concentrations of sulfur and ferrous iron. Such a titer is high enough to economically compete with the traditional chemical production process. Fungi Filamentous fungi are widely used for the commercial production of organic acids, for example, 1 billion pounds of citric acid are produced per year with a market value of US$1.4 billion. Citric acid is produced via the Embden-Meyerhof pathway and the first step of the tricarboxylic acid cycle; the control of the process involves the inhibition of phosphofructokinase by citric acid. The commercial process uses Aspergillus niger in media deficient in iron and manganese. A high level of citric acid production is also associated with a high intracellular concentration of fructose 2,6-biphosphate, an activator of glycolysis. Other factors contributing to high citric acid production are the inhibition of isocitrate dehydrogenase by citric acid and the low optimum pH (1.7–2.0). Higher pH values (e.g. 3.0) lead to the production of
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oxalic and gluconic acids, instead of citric acid. The low pH inactivates glucose oxidase, which would normally yield gluconic acid. In approximately 4–5 days, the major proportion (80%) of the sugar is converted to citric acid, with titers reaching about 100 g l21. Alternative processes have been developed for the production of citric acid by Candida yeasts, especially from hydrocarbons. Such yeasts are able to convert n-paraffins to citric and isocitric acids in extremely high yields [150–170% (w/w) of substrate used]; titers as high as 225 g l21 have been reached. Alcohol Ethyl alcohol is a primary metabolite produced by fermentation of sugar, or a polysaccharide that can be depolymerized to a fermentable sugar. Saccharomyces cerevisiae is used for the fermentation of hexoses, whereas Kluyveromyces fragilis or Candida species can be used if lactose or a pentose, respectively, is the substrate. Under optimum conditions, approximately 10–12% ethanol by volume is obtained within five days. Such a high concentration slows down growth and the fermentation ceases. With special yeasts, the fermentation can be continued to produce alcohol concentrations of 20% by volume, but these concentrations are attained only after months or years of fermentation. At present, all beverage alcohol is made by fermentation. Industrial ethanol is mainly manufactured by fermentation, but some is produced from ethylene by the petrochemical industry. Bacteria such as clostridia and Zymomonas are being re-examined for ethanol production after years of neglect. Clostridium thermocellum, an anaerobic thermophile, can convert waste cellulose (i.e. biomass) and crystalline cellulose directly to ethanol. Other clostridia produce acetate, lactate, acetone and butanol, and will be used to produce these chemicals when the gobal petroleum supplies begin to become depleted. Fuel ethanol produced from biomass would provide relief from air pollution caused by the use of gasoline and would not contribute to the greenhouse effect. E. coli has been converted into an excellent ethanol producer (43% yield, v/v) by recombinant DNA techniques. Bioconverting-organisms In addition to the multireaction sequences of fermentations, microorganisms are extremely useful in carrying out biotransformation processes in which a compound is converted into a structurally related product by one or a small number of enzymes contained in the cells5. Bioconverting-organisms are known for practically every type of chemical reaction. The reactions are stereospecific; the ultimate in specificity is exemplified by steroid bioconversions. Bioconversions are characterized by extremely high yields, approximately 90–100%. Other attributes include mild reaction conditions and the coupling of reactions, using a microorganism containing several enzymes working in series. Secondary metabolites Microbially produced secondary metabolites6 are extremely important for health and nutrition. As a group that includes antibiotics, other medicinals, toxins, biopesticides and animal and plant growth factors, they
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have tremendous economic importance. Secondary metabolites have no function in the growth of the producing cultures (although, in nature, they are essential for the survival of the producing organism), are produced by certain restricted taxonomic groups of organisms and are usually formed as mixtures of closely related members of a chemical family. Antibiotics The best-known group of the secondary metabolites are the antibiotics7. Their targets include DNA replication (actinomycin, bleomycin and griseofulvin), transcription (rifamycin), translation by 70-S ribosomes (chloramphenicol, tetracycline, lincomycin, erythromycin and streptomycin), transcription by 80-S ribosomes (cyclohexamide), transcription by 70- and 80-S ribosomes (puromycin and fusidic acid), cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin and vancomycin) and cell membranes (surfactants including: polymyxin and amphotericin; channelforming ionophores, such as linear gramicidin; and mobile carrier ionophores, such as monensin). Since 1940, there has been a virtual explosion of new and potent antibiotic molecules that have been of great use in medicine, agriculture and basic research. In 1996, the antibiotic market was composed of 160 antibiotics and amounted to a world market value of ~US$23 billion. The search for new antibiotics continues, in order to: combat evolving pathogens, naturally resistant bacteria and fungi, and previously susceptible microbes that have developed resistance; improve pharmacological properties; combat tumors, viruses and parasites; and discover safer, more potent and broaderspectrum compounds. In the search for new antibiotics, many of the new products are made chemically by modification of natural antibiotics via semisynthesis. Antibiotics are used not only for chemotherapy in human and veterinary medicine, but also for growth promotion in farm animals and for the protection of plants. Non-antibiotic agents In nature, secondary metabolites are important to the organisms that produce them, functioning as: (1) sex hormones; (2) ionophores; (3) competitive weapons against other bacteria, fungi, amoebae, insects and plants; (4) agents of symbiosis; and (5) effectors of differentiation. For years, most pharmaceuticals that were used for non-infectious diseases were strictly synthetic products8. Similarly, most therapeutics for nonmicrobial parasitic diseases in animals (e.g. coccidiostats and antihelminthics) came from the screening of synthesized compounds followed by molecular modification. Despite the testing of thousands of synthetic compounds, only a few promising structures were found. As new lead compounds became more and more difficult to find, microbial broths filled the void and microbial products increased in importance in the therapy of non-microbial diseases9. Today, microbially produced polyethers such as monensin, lasalocid and salinomycin dominate the coccidiostat market and are also the chief growth promoters in use for ruminant animals. The avermectins, another group of streptomycete products with a market of more than US$1 billion per year, have high activity against helminths and arthropods. TIBTECH JANUARY 2000 (Vol. 18)
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Many microbial products with important pharmacological activities were discovered by screening for inhibitors using simple enzymatic assays. One huge success has been the statins, including lovastatin (also known as mevinolin) and pravastatin: fungal products that are used as cholesterol-lowering agents in humans and animals. In its hydroxy acid form, lovastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from liver. Other wellknown enzyme inhibitors include: clavulanic acid, a penicillinase-inhibitor that protects penicillin from inactivation by resistant pathogens; and acarbose, a natural inhibitor of intestinal glucosidase, which is produced by an actinomycete of the genus Actinoplanes. Acarbose decreases hyperglycemia and triglyceride synthesis in adipose tissue, the liver and the intestinal wall of patients suffering from diabetes, obesity and type IV hyperlipidemia. Biopesticides Also in commercial or near-commercial use are biopesticides, including biofungicides (e.g. kasugamycin, polyoxins), bioinsecticides (nikkomycin, spinosyns), bioherbicides (bialaphos), antihelminthics (avermectin), coccidiostats, ruminant-growth promoters (monensin, lasalocid, salinomycin), plant-growth regulators (gibberellins), immunosuppressants for organ transplants (cyclosporin A, FK-506, rapamycin), anabolic agents in farm animals (zearelanone), uterocontractants (ergot alkaloids) and antitumor agents (doxorubicin, daunorubicin, mitomycin, bleomycin). Tropophase and idiophase In batch culture, most secondary metabolite processes have a distinct growth phase (trophophase) followed by a production phase (idiophase). In other fermentations, the two phases overlap; the timing depends on the nutritional environment presented to the culture, the growth rate, or both. A delay in antibiotic production until after trophophase helps the producing organism because the microbe is sometimes sensitive to its own antibiotic during growth. Resistance mechanisms that develop in producing microorganisms include enzymatic modification of the antibiotic, alteration of the cellular target of the antibiotic and decreased uptake of the excreted antibiotic. Directed biosynthesis The manipulation of the culture media in any development program often involves the testing of hundreds of additives as possible limiting precursors of the desired product. Occasionally, a precursor that increases production of the secondary metabolite is found. The precursor may also direct the fermentation towards the formation of one specific desirable product: this is known as directed biosynthesis. Examples of directed biosynthesis include the use of phenylacetic acid in the fermentation of benzylpenicillin, and specific amino acids in the production of actinomycins and tyrocidins. Stimulatory precursors include: methionine, as an inducer in cephalosporin C formation; valine, in tylosin production; and tryptophan for ergot-alkaloid production. In many fermentations, however, precursors show no activity because their syntheses are not rate-limiting. In such cases, TIBTECH JANUARY 2000 (Vol. 18)
screening of additives has often revealed dramatic effects, both stimulatory and inhibitory, of non-precursor molecules on the production of secondary metabolites. These effects are usually due to the interaction of these compounds with the regulatory mechanisms existing in the fermentation organism. Antibiotic biosynthesis ends via the decay of antibiotic synthetases or because of feedback inhibition and repression of these enzymes. Because the regulatory mechanisms are genetically determined, mutations have had a major effect on the production of secondary metabolites. Indeed, it is the chief factor responsible for the 100–1000-fold increases obtained in the production of antibiotics from their initial discovery to the present time. These tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by random mutagenesis and screening for higher-producing microbial strains. Mutation has also served to: (1) shift the proportion of metabolites produced in a fermentation broth to a more favorable distribution; (2) elucidate the pathways of secondary metabolism; and (3) yield new compounds. Modern microbial biotechnology Modern biotechnology is now over 25 years old10. In 1972, the birth of recombinant DNA technology propelled biotechnology to new heights and led to the establishment of a new industry. In addition to recombinant DNA technology, modern microbial biotechnology encompasses fermentation, microbial physiology, high-throughput screening for novel metabolites and strain improvement, bioreactor design and downstream processing, cell immobilization (enzyme engineering), cell fusion, metabolic engineering, bioreactor design, downstream processing, in vitro mutagenesis (protein engineering) and directed evolution of enzymes (applied molecular evolution). Recombinant microorganisms The revolutionary exploitation of microbial genetic discoveries in the 1970s, 1980s and 1990s depended heavily upon the solid structure of industrial microbiology, described above. The major microbial hosts for production of recombinant proteins are E. coli11, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorpha and Aspergillus niger. The use of recombinant microorganisms (Fig. 1) provided the techniques and experience necessary for the successful application of higher organisms, such as mammalian and insect cell culture, and transgenic animals and plants as hosts for the production of glycosylated recombinant proteins. Progress The progress in biotechnology has been truly remarkable. Within four years of the discovery of recombinant DNA technology, genetically engineered bacteria were making human insulin and human growth hormone. This led to an explosion of investment activity in new companies, mainly dedicated to innovation via genetic approaches. Newer companies entered the scene in various niches such as biochemical engineering and downstream processing. Today, biotechnology in the USA is represented by some 1300 companies with revenues of US$19.6 billion, of which sales represent US$13.4 billion, and there are
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was that of hepatitis B virus surface antigen produced in yeast. The great contribution made by recombinant vaccines is the elimination of the tragic problems associated with conventional vaccines. Through reversion of the attenuated pathogen, some individuals receiving the conventional vaccine not only failed to be protected, but also came down with the disease. Combinatorial biosynthesis As mentioned previously, recombinant DNA techniques have made a significant impact on the production of vitamins, amino acids, nucleotides, bioconversion and secondary metabolites. Most microbial biosynthetic pathways are encoded by clustered genes, which facilitates the transfer of an entire pathway in a single manipulation. Even in fungi, pathway genes are sometimes clustered, such as the penicillin genes in Penicillium or the aflatoxin genes in Aspergillus. For the discovery of new or modified secondary products, recombinant DNA techniques are being used to introduce genes for the synthesis of one product into producers of other antibiotics or into non-producing strains (combinatorial biosynthesis).
Figure 1 Escherichia coli: the workhorse of modern microbial biotechnology. (Electron micrograph taken by Erika Hartweig, bar = 1 mm.)
approximately 153 000 employees. The number of biotechnology companies in Canada reached 282 in 1998, employing 10 000 workers and with revenues of approximately US$1.1 billion. Japan’s biotechnology sales were approximately US$10 billion, mainly by established pharmaceutical, food and beverage companies. European biotechnology moved rapidly in the 1990s, after years of lagging behind and, in 1998, 1178 biotechnology companies existed with 45 000 employees, and revenues of US$3.7 billion. The major thrust of recombinant DNA technology has been in the area of rare mammalian peptides, such as hormones, growth factors, enzymes, antibodies and biological response modifiers12. Among those genetically engineered products that have been approved for use in the USA are human insulin, human growth hormone, erythropoietin, antihemophelia factor, granulocyte-colony stimulating factor, granulocytemacrophage-colony stimulating factor, epidermal growth factor and other growth factors, interleukin-2, a-, b- and g-interferons, and bovine somatotropin. Vaccines Vaccine production is another important part of the new technology; the first subunit vaccine on the market
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Enzyme production The production of enzymes by fermentation was an established business before modern microbial biotechnology. However, recombinant DNA methodology was so perfectly suited to the improvement of enzymeproduction technology that it was almost immediately used by companies involved in manufacturing enzymes. Industrial enzymes have now reached an annual market of US$1.6 billion. Important enzymes are proteases, lipases, carbohydrases, recombinant chymosin for cheese manufacture and recombinant lipase for use in detergents. Recombinant therapeutic enzymes already have a market value of over US$2 billion, being used for thromboses, gastrointestinal and rheumatic disorders, metabolic diseases and cancer. They include tissue plasminogen activator, human DNAase and Cerozyme. Agriculture Industrial microbiology through genetic engineering and its associated disciplines has brought about a revolution in agriculture. Two bacteria have had a major influence: Agrobacterium tumefaciens, a bacterium that normally produces crown gall tumors on dicotyledonous plants; and Bacillus thuringiensis, an insecticidal bacterium. The tumor-forming genes of A. tumefaciens are present on its tumor-inducing (Ti) plasmid, along with genes directing the plant to form opines (nutritional factors required by the bacterium that it cannot produce by itself ). The Ti vector has been exceedingly valuable for introducing foreign genes into dicotyledonous plants for production of transgenic plants. However, the Ti plasmid is not very successful for transferring genes into monocotyledonous plants, a problem bypassed by, for example, the development of a particleacceleration gun, which shoots DNA-coated metal particles into plant cells. The activity of the insecticidal bacterium, B. thuringiensis, is caused by its crystal protein produced during sporulation. Crystals and spores have been applied to plants for many years to protect them against lepidopteran insects. B. thuringiensis preparations are highly potent, approximately 300 times more active TIBTECH JANUARY 2000 (Vol. 18)
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on a molar basis than synthetic pyrethroids and 80 000 times more active than organophosphate insecticides. In the modern biotechnology era, plants resistant to insects have been produced by expressing forms of the B. thuringiensis toxin gene in the plant. Recently developed bioinsecticides include insect viruses, such as baculoviruses, that are engineered to produce arthropod toxins. Transgenic plants, resistant to herbicides, are also available, as are virus-resistant plants produced by expressing viral-coat-protein genes in plants. Interestingly, chemical pesticides against plant viruses were never available. Conclusion Although most of the early promises of biotechnology have been achieved, major challenges remain. We must use our brains, technology, drive and dedication to solve the problems of evolving diseases (e.g. AIDS), established diseases (cancer and parasitic infection), antibioticresistance development and environmental pollution, by converting urban, industrial and agricultural wastes into resources such as liquid fuel. These efforts will require continued interaction between different disciplines, major support by governments and international agencies, as well as an understanding and supportive public. References 1 Demain, A.L. (1990) Achievements in microbial technology. Biotechnol. Adv. 8, 291–301
2 Demain, A.L. (1988) Contributions of genetics to the production and discovery of microbial pharmaceuticals. Pure Appl. Chem. 60, 833–836 3 Jetten, M.S. and Sinskey, A.J. (1995) Recent advances in the physiology and genetics of amino acid-producing bacteria. Crit. Rev. Biotechnol. 15, 73–103 4 Sahm, H. et al. (1995) Metabolic design in amino acid producing bacterium Corynebacterium glutamicum. FEMS Microbiol. Rev. 16, 243–252 5 Kieslich, K. (1997) Biotransformations. In Fungal Biotechnology (Anke, T., ed.), pp. 297–399, Chapman & Hall 6 Demain, A.L. (1992) Microbial secondary metabolism: a new theoretical frontier for academia, a new opportunity for industry. In Secondary Metabolites: Their Function and Evolution (Chadwick, D.J. and Whelan, J., eds), pp. 3–23, John Wiley & Sons 7 Strohl, W.R. (1997) Biotechnology of Antibiotics, 2nd edn, Marcel Dekker 8 Demain, A.L. (1996) Fungal secondary metabolism: regulation and functions. In A Century of Mycology (Sutton, B., ed.), pp. 233–254, Cambridge University Press 9 Demain, A.L. (1998) Microbial natural products: alive and well in 1998. Nat. Biotechnol. 16, 3–4 10 Cohen, S.N. (1979) The transplantation and manipulation of genes in microorganisms. The Harvey Lectures 74, 173–204 11 Swartz, J.R. (1996) Escherichia coli recombinant DNA technology. In Escherichia coli and Salmonella: Cellular and Molecular Biology 2nd edn (Neidhardt, F.C., ed.), pp. 1693–1711, American Society of Microbiology (ASM) Press 12 Demain, A.L. et al. (1994) Contributions of recombinant microbes and their potential. In Recombinant Microbes for Industrial and Agricultural Applications (Murooka, T. and Imanaka, T., eds), pp. 27–46, Marcel Dekker
Drug discovery in the new millennium: the pivotal role of biotechnology Graham J. Boulnois Biotechnology has made, and will continue to make, a major contribution to the health of mankind by providing new insights into disease and acting as a pivotal enabler for the drug-discovery process. The available techniques are diverse and changing rapidly: selecting and integrating the best approach is the key to success.
odern medicines have improved the life of humankind, and the past few decades have seen enormous advances in our ability to treat, manage and prevent a large number of diseases. Successful therapies range from the treatment of acute infections with powerful antibiotics and the availability of anaesthetics for surgery and management, through risk reduction in cardiovascular disease to the prevention of a range of infectious diseases via vaccination. Biotechnology has already played a major role in modern medicine discovery, delivering a range of new treatments and vaccines in its own right, and gene therapy is just round the corner. Despite these impressive
M
G.J. Boulnois ([email protected]) is at AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK SK10 4TG. TIBTECH JANUARY 2000 (Vol. 18)
advances, there are many diseases for which treatments are lacking or are imperfect, and we are a long way from eradicating or even reducing the prevalence of many debilitating conditions. In short, the opportunities for new treatments is huge. We have also seen a change in the way new medicines are discovered, driven by biotechnology in general but particularly by molecular biology. The reliance on testing new synthetic organic molecules in animals or in whole-organ preparations, as an early part of the discovery process, has changed to a molecular target approach in which in vitro screening of compounds against purified, recombinant proteins or genetically modified cell lines is carried out with a high throughput. This change has come about as a consequence of better and ever-improving knowledge of the molecular basis
0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01393-1
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