The Advent of Modern Microbiology: Stunning Achievements of Industrial Microbiology

    Stunning Achievements of Industrial Microbiology

    Microorganisms produce valuable polymers, such as proteins, nucleic acids, and polysaccharides, as well as many smaller molecules useful in human and animal health

    Arnold L. Demain

    David Perlman of the University of Wisconsin often reminded his students and colleagues that, "If you take care of your microbial friends, they will take care of your future." How right he was. Microorganisms are important because they produce things we value, including materials such as proteins, nucleic acids, carbohydrate polymers, cells, and also smaller molecules which we usually divide into metabolites essential for vegetative growth (primary) and nonessential (secondary) metabolites.

    The unique importance of industrial microbiology resides in five important characteristics of microorganisms:

    • a high ratio of surface area to volume, which facilitates the rapid uptake of nutrients required to support high rates of metabolism and biosynthesis;
    • a tremendous variety of reactions which microorganisms are capable of carrying out;
    • adaptability to a large array of different environments, including the laboratory flask and factory fermentor, and growth conditions;
    • ease of genetic manipulation to increase productivity and to modify those end products; and
    • ability to make specific enantiomers, in cases where conventional chemical synthesis yields a mixture of active and inactive enantiomers.

    The power of the microbial culture in the competitive world of commercial synthesis can be appreciated by the fact that even simple molecules, such as l-glutamic acid, are made by fermentation rather than by chemical synthesis. Although a few products have been temporarily lost to chemical synthesis (e.g., acetone and butanol), most natural products are made by fermentation technology because they contain so many centers of asymmetry.

    Industrial Processes Overcome Natural Regulatory Mechanisms

    Although microbes make an amazing array of valuable products, regulatory mechanisms limit cells to producing only tiny amounts needed for their own benefit.

    Meanwhile, industrial microbiologists typically desire a "wasteful" strain which will overproduce a particular compound that can be isolated and purified. During the screening stage, microbiologists search for organisms with weak regulatory mechanisms. Subsequently a development program to improve titers involves modifying culture conditions, finding mutants, or using recombinant DNA technology to boost production.

    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. Thousand-fold increases have been recorded for small metabolites. Of course, the higher the specific level of production, the simpler it is to isolate the product. Consider the case of Ashbya gossypii, which makes more than 40,000 times more riboflavin than it needs, or Pseudomonas denitrificans, which produces a 100,000-fold excess of vitamin B12. The original Oxford strain of Penicillium notatum produced 5 mg of penicillin per liter; today's strains make more than 60,000 mg per liter, a figure higher than the dry weight of the cells in the fermentor!

    Recombinant DNA and Biotechnology Bring Many Changes

    The revolutionary exploitation of microbial genetic discoveries did not take place in a vacuum but heavily depended upon the solid structure of industrial microbiology. Louis Pasteur was one of the first practitioners when he solved the problem of bacterial contaminants responsible for the acidification of the famous wines of Bordeaux. He was hailed as a hero by the wine makers.

    Early in this century, E. Wildiers published the first description of a microbial growth factor, opening the field of vitamin research. He found that a water-soluble extract of yeast contained a compound that was required for the growth of yeast. The material was later determined to be a B vitamin. Shortly after, in 1904, Franz Schardinger isolated aerobic bacilli which produce acetone, ethanol, and acetic acid. Chaim Weizmann, using the knowledge of Pasteur's discovery that yeast ferments sugar, used Clostridium acetobutylicum to produce acetone and butyl alcohol. He received a British patent in 1915. These chemicals were essential to the British munitions program during World War I.

    After World War I, industrial microbiology split into two streams; one, after Fleming's momentous discovery of penicillin in 1929, was directed at discovery and industrial production of antibiotics. Another group attempted to genetically manipulate bacteria and yeasts to increase production of specific industrial and commercial chemicals. For example, J. N. Currie in 1917 discovered how to produce citric acid in large quantities from the mold Aspergillus niger by employing a growth-limiting medium rich in iron. Later, in 1930, Henning Karstrom began to identify the phenomena of enzyme a daptation and of constitutive synthesis, in which synthesis of an enzyme either is increased in response to the presence of the substrate in the environment or is independent of the growth medium. His work was based on studies of carbohydrate metabolism in gram-negative enteric bacteria. Mary Shorb's Lactobacillus lactis assay was employed at Merck & Co. to guide purification and crystallization of vitamin B12 from Streptomyces griseus. B12 was applied to the treatment of pernicious anemia in humans, and as the animal protein factor, for promotion of growth in farm animals. In the mid-1950s, Shukuo Kinoshita, S. Udaka, and M. Shimono discovered that bacteria can be used to produce monosodium glutamate. This led to a new industry: the microbial production of amino acids for human and animal nutrition as well as for food flavoring.

    The flowering of the field occurred subsequent to the recombinant DNA discoveries made in 1972-73 in the laboratories of Paul Berg, Stanley Cohen, and Herbert Boyer at Stanford University and the University of California at San Francisco. Research on recombinant microorganisms has helped to provide the technology and experience needed to produce recombinant proteins and to develop mammalian cell culture techniques, insect cell culture, and transgenic animals and plants as hosts for producing useful entities, including glycosylated proteins. A major new activity is applied molecular evolution (DNA shuffling) for the improvement of proteins with respect to enzyme activity, specificity, folding, and other properties.

    Although production of enzymes by fermentation was an established business prior to the age of modern biotechnology, recombinant DNA methodology was so perfectly suited to the improvement of enzyme production technology that it was immediately utilized by companies manufacturing enzymes. Industrial enzymes have now reached an annual market of $1.6 billion, of which proteases used in detergents account for $200 million. Other important enzymes are recombinant chymosin for cheese manufacture and recombinant lipase for use in detergents.

    Within four years of the discovery of recombinant DNA, genetically engineered bacteria were also making human insulin and human growth hormone. Within 15 years after recombinant DNA technology developed, about 440 biotechnology companies and 70 large pharmaceutical, chemical, and energy corporations in the United States were devoting aggregate resources of $1.5 to 2 billion to biotechnology per year. U.S. biotechnology is now represented by 1,300 companies, with revenues of $17-18 billion and 140,000 employees.

    In the past, pharmaceutical research mainly aimed at acute problems, such as bacterial and fungal infections, symptomatic relief of pain, insomnia, hypertension, and other medical disorders. Today, research focuses on chronic or complex acute problems such as atherosclerosis, degenerative disorders of the central nervous and musculoskeletal systems, autoimmune and chronic inflammation, and cancer. In addition, immunosuppression (for organ transplants) and viral infections are current targets. Molecular biology has become the major driving force in pharmaceutical research.

    Microorganisms Are the Commercial Source of Primary Metabolites

    Penicillium spp
    Mucor circenelloides

    One major segment of the field supports microbially based manufacturing of a wide variety of primary and secondary metabolites. Primary metabolites are the small molecules of all living cells that are intermediates or end products of the pathways of intermediary metabolism, or are building blocks for essential macromolecules, or are converted into coenzymes. The most industrially important are the amino acids, nucleotides, vitamins, solvents, and organic acids. It is not unexpected that amino acids and vitamins are used in human and animal nutrition, that ethanol, acetone, and butanol are used as fuels and/or solvents, and that citric and acetic acids are used as acidulants. However, some primary metabolites are used in novel ways: the sodium salts of glutamic, 5'-inosinic and 5'- guanylic acids as flavor enhancers, sodium gluconate as a sequestering agent to prevent the deposition of soap scums on cleaned surfaces, and fumarate in the manufacture of polyester resins.

    In amino acid production, feedback regulation is bypassed by isolating auxotrophic mutants and partially starving them of their requirement. A second means is to produce mutants resistant to a toxic analog of the desired metabolite, i.e., an antimetabolite. Combinations of auxotrophic and antimetabolite resistance mutations are common in primary metabolite- producing microorganisms.

    Perhaps the most significant primary metabolite is l-glutamic acid, the major amino acid of commerce. About 1.2 billion pounds of monosodium glutamate are made annually by fermentation using various species of the genera Corynebacterium and Brevibacterium. The major route of glutamate production from glucose is via the Embden-Meyerhof pathway and the early steps of the tricarboxylic acid cycle. Thus, alpha-ketoglutarate, which would normally be converted to succinyl-coenzyme A in the cycle, is instead reductively aminated to glutamate by glutamate dehydrogenase.

    Normally, glutamic acid overproduction would not occur because of feedback regulation. However, a phospholipid-deficient cytoplasmic membrane enables cells to pump glutamate into the medium, thus allowing biosynthesis to proceed unabated. Also required for effective production are high levels of carbon and energy sources as well as growth inhibition. The actual excretion is carried out by a specific efflux system involving a carrier that is dependent upon membrane potential.

    Microorganisms Are Also Important for Vitamin Production

    Riboflavin (vitamin B2) is produced commercially by both fermentation and chemical synthesis. Riboflavin overproducers include two yeast-like molds, Eremothecium ashbyii and Ashbya gossypii, which synthesize riboflavin in concentrations greater than 20 g/liter.

    The biochemical key to riboflavin overproduction appears to involve resistance to the repressive effects of iron. Ferrous ion severely inhibits riboflavin production by low and moderate overproducers, such as clostridia and Candida spp., but has no inhibitory action against Eremothecium ashbyii and Ashbya gossypii. In normal microorganisms, it appears that iron represses almost all of the riboflavin biosynthetic enzymes, whereas riboflavin or a derivative inhibits the first enzyme of the pathway, GTP cyclohydrolase II. New processes using Candida species or recombinant Bacillus subtilis strains have recently been developed which yield 20 to 30 g/liter.

    Vitamin B12 is industrially produced by Propionibacterium shermanii or Pseudomonas denitrificans. The key to the fermentation is avoidance of feedback repression by vitamin B12. The early stage of the Propionibacterium 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. Then the culture is aerated and dimethylbenz-imidazole is added, converting cobinamide to the vitamin.

    In production of biotin, acetyl-coenzyme A carboxylase biotin holoenzyme synthetase causes feedback repression, with biotin 5-adenylate acting as corepressor. Modified strains of Serratia marcescens produce 600 mg of biotin per liter in the presence of high concentrations of sulfur and ferrous iron. Such a titer is high enough to compete with chemical production.

    Organic Acid and Alcohol Production

    Filamentous fungi are widely used for the commercial production of organic acids. About 1 billion pounds of citric acid are produced per year, with a market value of $1.4 billion. This organic acid is produced via the Embden-Meyerhof pathway and the first step of the tricarboxylic acid cycle. The major control of the process involves the inhibition of phosphofructokinase by citric acid. The commercial process employs Aspergillus niger in media deficient in iron and manganese. Citric acid is easily assimilated and palatable and exhibits low toxicity. Consequently, it is widely used in the food and pharmaceutical industries. It is employed as an acidifying and flavor-enhancing agent, as an antioxidant for inhibiting rancidity in fats and oils, as a buffer in jams and jellies, and as a stabilizer in a variety of foods.

    Ethyl alcohol is a primary metabolite that can be produced by fermentation of a sugar or a polysaccharide that can be depolymerized to a fermentable sugar. Yeasts are preferred for these fermentations, but the species used depends on the substrate employed. Saccharomyces cerevisiae is employed for the fermentation of hexoses, whereas Kluyveromyces fragilis or Candida species may be utilized if lactose or a pentose, respectively, is the substrate.

    Under optimum conditions, approximately 10 to 12% ethanol by volume is obtained within 5 days. Such a high concentration slows down growth and the fermentation ceases. With special yeasts, the fermentation can produce alcohol concentrations of 20% by volume, but only after extended fermentations. Although all beverage alcohol is made by fermentation, some ethanol for industrial uses is made by the petrochemical industry from ethylene.

    Bacteria such as clostridia and Zymomonas are being reexamined for ethanol production after years of neglect. For instance, Clostridium thermocellum, an anaerobic thermophile, converts waste cellulose directly to ethanol. Other clostridia produce acetate, lactate, acetone, and butanol. Because of the elimination of lead from gasoline, ethanol is being substituted as a blend to raise gasoline's octane rating.

    Escherichia coli has been converted into an excellent ethanol producer (43% [vol/vol]) by recombinant DNA technology. Alcohol dehydrogenase and pyruvate decarboxylase genes from Zymomonas mobilis were inserted in E. coli and became the dominant system for NAD regeneration. Ethanol represents over 95% of the fermentation products in the genetically engineered strain.

    Microorganisms Important for Making Secondary Metabolites

    In nature, secondary metabolites are important for the organisms that produce them, functioning as (i) sex hormones, (ii) ionophores, (iii) competitive weapons against other bacteria, fungi, amoebae, insects and plants, (iv) agents of symbiosis, and (v) effectors of differentiation. For years, the major pharmaceuticals (such as hypertensive and anti-inflammatory agents) used for noninfectious diseases were strictly synthetic products. Similarly, major therapeutics for nonmicrobial parasitic diseases in animals (e.g., coccidiostats and antihelmintics) 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 uncovered. As new lead compounds became more and more difficult to find, microbial broths filled the void and microbial products increased in importance.

    This group includes antibiotics, other medicinals, toxins, pesticides, and animal and plant growth factors; they have tremendous economic importance. The best-known secondary metabolites are the antibiotics. This remarkable group of compounds forms a heterogeneous assemblage of biologically active molecules with different structures and modes of action.

    In batch or fed-batch culture, secondary metabolites are produced usually after growth has slowed down. They have no function in growth of the producing cultures (although they are essential for survival of the producing organism in nature), are produced by certain restricted taxonomic groups of organisms, and are usually formed as mixtures of closely related members of a chemical family.

    Recombinant DNA technology has improved the production of antibiotics. Even when the antibiotic biosynthetic pathway genes are chromosomal, they are often clustered, which facilitates transfer of an entire pathway in a single manipulation. However, the genes coding for cephalosporin biosynthesis in Cephalosporium spp. are distributed among different chromosomes.

    For the discovery of new or modified products, recombinant DNA techniques can be used to introduce genes encoding antibiotic synthases into producers of other antibiotics or into nonproducing strains to obtain modified or hybrid antibiotics (combinatorial biosynthesis). For instance, gene transfer from a streptomycete strain producing the isochromanequinone antibiotic actinorhodin into a strain producing granaticin, dihydrogranaticin, and mederomycin (which are also isochromanequinones) led to the discovery of two new antibiotic derivatives, mederrhodin A and dihydrogranatirhodin. Since then, many novel polyketide secondary metabolites have been obtained by cloning DNA fragments from one polyketide producer into various strains of other streptomycetes.

    Since 1940, we have witnessed a virtual explosion of new and potent antibiotic molecules which have been of great use in medicine, agriculture, and basic research. In 1996, the antibiotic market utilized 160 antibiotics and amounted to $23 billion. About 6,000 antibiotics have been described, 4,000 from actinomycetes alone, and they still are being discovered at a rate of about 500 per year. From 1990 to 1994, over 1,000 new secondary metabolites were characterized from actinomycetes alone. Some species are amazing in their ability to produce large numbers of antibiotics; e.g., strains of Streptomyces hygroscopicus make a total of almost 200 antibiotics. 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; to improve pharmacological properties; to combat tumors, viruses, and parasites; and to discover safer, more potent, and broader-spectrum compounds.

    Secondary Metabolites with Other Pharmacologic Activities

    Many microbial products with important pharmacological activities were discovered by screening for inhibitors using simple enzymatic assays. One huge success has been lovastatin, a fungal product which acts as a cholesterol-lowering agent in animals. Lovastatin is produced by Aspergillus terreus. In its hydroxy acid form (mevinolinic acid), lovastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl- coenzyme A reductase from liver.

    Another enzyme inhibitor on the market is acarbose, a natural inhibitor of intestinal glucosidase, which is produced by an actinomycete of the genus Actinoplanes. It decreases hyper- glycemia and triglyceride synthesis in adipose tissue, liver, and the intestinal wall of patients suffering from diabetes, obesity, and type IV hyperlipidemia.

    Also in commercial or near-commercial use are biopesticides including fungicides (e.g., kasugamycin, polyoxins), insecticides (Bacillus thuringiensis crystals, nikkomycin, spinosyns), herbicides (bialaphos), antihelmintics and 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).

    Many of these compounds were first isolated as poor or toxic antibiotics (e.g., monensin, cyclosporin, rapamycin) or as mycotoxins (ergot alkaloids, gibberellins, zearelanone) before they were put to work for our benefit. Following in their footsteps are a large number of secondary metabolites with potent activities which are currently under investigation. Indeed, antibiotic activity is merely the tip of the iceberg, a mere scratching at the surface of the potential of microbial activity.

    Professor Jackson W. Foster of the University of Texas once said, "Never underestimate the power of the microbe." Indeed, industrial microbiologists never have.

    SUGGESTED READING

    Chater, K. F. 1990. The improving prospects for yield increase by genetic engineering in antibiotic-producing streptomycetes. Bio/Technology 8:115-121.

    Demain, A. L. 1990. Achievements in microbial technology. Biotechnol. Adv. 8:291-301.

    Demain, A. L. 1992. Microbial secondary metabolism: a new theoretical frontier for academia, a new opportunity for industry, p. 3-23. In D. J. Chadwick and J. Whelan (ed.), Secondary metabolites: their function and evolution. John Wiley & Sons, New York.

    Demain, A. L., A. Kimura, and A. Shinmyo. 1994. Contributions of recombinant microbes and their potential, p. 27-46. In T. Murooka, and T. Imanaka (ed.), Recombinant microbes for industrial and agricultural applications. Marcel Dekker, New York.

    Demain, A. L. 1996. Fungal secondary metabolism: regulation and functions, p. 233-254. In B. Sutton (ed.), A century of mycology. Cambridge University Press, Cambridge.

    Demain, A. L. 1998. Microbial natural products: alive and well in 1998. Nature Biotechnol. 16:3-4. Jetten, M. S. M., and A. J. Sinskey. 1995. Recent advances in the physiology and genetics of amino acid-producing bacteria. Crit. Rev. Biotechnol. 15:73-103.

    Kuninaka, A. 1996. Nucleotides and related compounds, p. 561-612. In H.-J. Rehm, G. Reed, A. P&Mac197;hler, and P. Stadler (ed.), Biotechnology, 2nd ed., Vol. 6. VCH Verlagsgesellschaft, Weinheim.

    Sahm, H., L. Eggeling, B. Eikmanns, and R. Kr-mer. 1995. Metabolic design in amino acid producing bacterium Corynebacterium glutamicum. FEMS Microbiol. Rev. 16:243-252.

    Strohl, W. R. 1997. Biotechnology of antibiotics, 2nd edition. Marcel Dekker, New York.

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