General Microbiology - Schlegel H. 1987

Incomplete Oxidations
Formation of Secondary Metabolites

The discovery of penicillin and Other Antibiotics gave rise to a vast new field of industrial microbiology. Bacteria and Fungi synthesize numerous compounds that, by analogy with similar substances of plant origin, are commonly referred to as secondary metabolites. Many of these compounds play a crucial role as therapeutics, growth stimulants, feed additives, and the like. As producers of secondary metabolites, microorganisms have acquired immense economic importance. The discovery and study of antibiotics, as well as The production of novel semisynthetic derivatives, have rendered an invaluable service to medicine. We cannot yet foresee all the possibilities that future research in this field will unlock, nor all the new Applications for microbial secondary metabolites. The generation and Selection of mutants (taking regulatory mechanisms into account) will significantly expand The Scope of microbial syntheses.

10.4.1 Production of Antibiotics

It was already known in the last century that both symbiotic and antagonistic relationships can exist between various microorganisms. The impetus for elucidating the material basis of antibiosis came from Fleming's observation in 1928 that a colony of the fungus Penicillium notatum inhibited the growth of staphylococci. The substance secreted by this fungus, which diffused into the Agar, was named penicillin. Since then, a multitude of substances with antibiotic activity have been isolated. Antibiotics are substances of biological origin capable of inhibiting Microbial growth even at low concentrations. A distinction is made between substances that arrest microbial growth (bacteriostatic, fungistatic) and those that destroy microorganisms outright (bactericidal, fungicidal, etc.).

Antibiotic producers. The synthesis of antibiotics is carried out primarily by fungi of the order Aspergillales, actinomycetes, and certain other bacteria. Streptomycetes rank first in the chemical Water/126.html">Diversity of the substances they synthesize. To date, more than 2,000 antibiotics have been thoroughly characterized, yet only about fifty are utilized as chemotherapeutic agents. The number of described antibiotic interactions is far greater, but many groups of microorganisms—including uncultivable or fastidious bacteria and lower fungi—remain insufficiently studied in this regard.

The Significance of antibiotics for the organisms that produce them. The question of what function antibiotics serve their producers in their natural habitat, the soil, remains unclear. The synthesis of antibiotics follows specialized biochemical pathways associated with Secondary METABOLISM. These pathways and the Enzymes that drive them are not essential for Cell growth and survival. If the genetic apparatus required for antibiotic synthesis were useless, it would represent metabolic ballast, and the Organism would have shed it in the course of evolution through respective deletions. Since nature evidently preserves only what is functional, antibiotics must be viewed as substances that afford their producers a selective advantage under natural conditions, i.e., in the soil (such as a competitive edge for a shared substrate). However, such antagonistic interactions are difficult to detect in the soil because antibiotics are produced in very small quantities; furthermore, they generally inhibit the growth of the producers themselves.

Gradually, the view is gaining ground that evolution can preserve seemingly unnecessary genetic material—even when it proves to be metabolic ballast under experimental conditions studied thus far. Apparently, nature is far more conservative than was assumed at the dawn of the molecular biology era. Today, antibiotics and other secondary metabolites, whose direct utility to the synthesizing Cells is difficult to discern, are metaphorically classed as metabolic "shavings" or products arising from the "playground" of metabolism. This example clearly illustrates that studying the secondary metabolism of bacteria, fungi, and plants is one of the most promising avenues for investigating the pathways of organic evolution.

Methods for detecting antibiotics. The first antibiotics were discovered serendipitously through The formation of growth inhibition zones. In Petri dishes densely seeded with a test organism (indicator bacteria), growth was absent around colonies of a fungus or streptomycete: the antibiotic diffusing from the colony into the agar produced clear halos within the confluent bacterial lawn (Fig. 10.4). Typical representatives of various microbial groups serve as indicator species (test objects) in such experiments. For a qualitative test of an antibiotic producer, it is sufficient to inoculate it in the center of an agar plate, with the indicator bacteria streaked radially outward (cross-streak method, Fig. 10.5). Following incubation, THE SPECTRUM OF antibiotic action is assessed by the degree of growth inhibition of the various indicator organisms. Antibiotics differ in their activity against Gram-positive and Gram-negative bacteria, Yeasts, dermatophytes, and other microorganisms.

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Fig. 10.4. The production of antibiotics by bacteria or fungi can be detected by the formation of growth-inhibition zones against indicator bacteria (Staphylococcus aureus) evenly distributed throughout the agar.

Fig. 10.5. Determining the spectrum of action of three antibiotics using the streak test. 1 - Staphylococcus aureus; 2 - Streptococcus; 3 - Escherichia coli; 4 - Pseudomonas aeruginosa; 5 - Candida albicans; 6 - Trichophyton rubrum.

A filter-paper disc saturated with a solution of the test antibiotic (approx. 10 µg of the antibiotic) is placed in the center of a Petri dish containing peptone-casein hydrolysate agar. Suspensions of the test organisms are applied with a platinum loop as radial streaks (one to six). Certain microorganisms fail to grow within the antibiotic diffusion zone. (Wallhäusser K. H., Schmidt H., Sterilisation, Desinfektion, Konservierung, Chemotherapie. Stuttgart: Thieme, 1967.)

The majority of antibiotics have been discovered through preliminary screening Procedures. Fig. 10.6 illustrates the complete workflow—from preparing a soil sample suspension to animal testing.

Quantitative determination. To quantitatively assess antibiotic activity, the agar diffusion assay (Fig. 10.7), the serial dilution method, and several other techniques are employed. For the diffusion assay, Petri dishes are filled to a specified depth with an agar medium containing a suspension of the test organism. The test antibiotic solutions are then introduced into the plates—either into wells, Glass or metal cylinders, or by applying filter-paper discs impregnated with the antibiotic onto the agar. In all cases of a positive reaction, a zone of growth inhibition surrounding the test organism becomes apparent after incubation. When constant experimental conditions are maintained (medium composition, agar layer thickness, inoculation density, incubation time, Temperature, etc.), the diameter of this zone is proportional to the logarithm of the antibiotic concentration (Fig. 10.7).

When using the serial dilution method, a twofold dilution series of the antibiotic is prepared in a nutrient broth inoculated with the test organism, and following incubation, the lowest antibiotic concentration that prevents visible growth (the minimum inhibitory concentration) is determined.

Specialized methods have been developed to establish the synergistic and antagonistic effects of various substances, as well as to investigate The impact of antibiotics on other organisms (Protozoa, worms, Algae, cell cultures, Viruses).

Fig. 10.6. Flowchart for antibiotic screening. (Wallhäusser K. H., Schmidt H., Sterilisation, Desinfektion, Konservierung, Chemotherapie. Stuttgart: Thieme, 1976.)

Fig. 10.7. Quantitative Assay of an antibiotic by the plate diffusion method. Filter-paper discs placed On the surface of the seeded agar medium contain varying amounts of the antibiotic. The diameter of the test organism's growth inhibition zone is proportional to the antibiotic concentration. (Zähner H., Biologie der Antibiotica. Berlin: Springer, 1965.)

Major medical antibiotics. Penicillin still holds first place among them, synthesized by Penicillium notatum, P. chrysogenum, and certain other fungi; Semisynthetic Penicillins have also been successfully produced (by cleaving natural penicillins into 6-aminopenicillanic acid, to which various side chains are subsequently attached chemically). The Mechanism of penicillin action on bacteria was discussed earlier (Section 2.2.3). Penicillin exhibits almost no toxicity to humans, only rarely causing allergic side effects. Many bacteria produce penicillinase, which cleaves the ß-lactam ring and inactivates penicillin. By reacting 6-aminopenicillanic acid with acyl chlorides, hundreds of different penicillins can be synthesized (Fig. 10.8). Many semisynthetic penicillins are resistant to penicillinase and, owing to their acid stability, can be administered orally.

Fig. 10.8. Action of the bacterial enzymes penicillinase and penicillin acylase on penicillin G. By substituting R in 6-aminopenicillanic acid with various radicals, semisynthetic penicillins such as phenethicillin, methicillin, ampicillin, and carbenicillin can be obtained.

Fig. 10.9. Structural formulas of cephalosporin C, streptomycin A, chloromycetin (chloramphenicol), tetracycline, and actinomycin D (actinomycin C1).

Cephalosporins are products derived from a fungus species, Cephalosporium. Cephalosporin C contains a ß-lactam ring and is structurally similar to penicillin (Fig. 10.9). By cleaving the side chain and subsequently attaching other side groups to the resulting 7-aminocephalosporanic acid, semi-synthetic cephalosporins (such as cephalothin and cephaloridine) can be produced, which are similar in their action to penicillin derivatives.

Streptomycin was first isolated from a culture of Streptomyces griseus, although it is also synthesized by several other Streptomyces species. The streptomycin molecule consists of three parts: N-methyl-L-glucosamine, methylpentose, and a diguanidine-substituted Inositol (Fig. 10.9). The success of streptomycin is due to its activity against a range of acid-fast and Gram-negative bacteria that are resistant to penicillin. However, streptomycin causes pronounced allergic reactions in patients. This antibiotic is also used in veterinary medicine and for controlling plant diseases.

Chloromycetin (chloramphenicol) was first discovered in cultures of Streptomyces venezuelae, but it can also be produced synthetically (Fig. 10.9). It is exceptionally stable and acts on many Gram-negative bacteria, including spirochetes, rickettsiae, and actinomycetes, as well as large viruses.

Tetracyclines are likewise metabolites of various streptomycetes (including Streptomyces aureofaciens). Chemically, they are closely related and share a naphthacene-based Structure (Fig. 10.9). The best known are chlortetracycline (aureomycin), oxytetracycline (terramycin), and tetracycline. Tetracyclines are characterized by a broad spectrum of activity and good tolerability.

Macrolides comprise antibiotics of diverse origins with relatively high molecular weights, characterized by a macrocyclic lactone ring (e.g., erythromycin, carbomycin A, picromycin).

Actinomycin was isolated in 1940 (the first of the antibiotics produced by streptomycetes). It is a mixture of several substances, united only by a phenoxazone chromophore. Various polypeptide chains serve as substituents (Fig. 10.9). Finally, mention should be made of the group of Polypeptide antibiotics (gramicidin S, polymyxins, bacitracin, ristocetin, etc.). In the polymyxin B molecule, a side chain is attached via a peptide bond to a ring of Seven Amino Acids (Fig. 10.10). Polypeptide antibiotics have a high affinity for The Plasma Membrane; consequently, they are equally toxic to both bacteria and eukaryotes and are not used clinically. Due to their ability to selectively transport ions across membranes, polypeptide antibiotics can be used for research purposes as ionophores (see Section 7.7). Valinomycin, for instance, facilitates The transport of potassium ions across the membrane. The molecule of this antibiotic includes a 12-membered ring (comprising valine, 2-hydroxyisovalerate, and lactate, among others). The structure of this ring is such that a potassium ion fits precisely within the internal space of the molecule. The valinomycin-K⁺ complex is lipophilic due to the presence of valine and valerate, and is easily transported across the lipid layer of the membrane. Therefore, The addition of valinomycin to a cell suspension leads to the loss of potassium ions by the cells.

Fig. 10.10. Polymyxin B. L-Dab - 2,4-diaminobutyric acid; L-leu - L-leucine; D-Phe - phenylalanine; L-Thr - Threonine; aliphatic side chain - 6-methyloctanoic acid.

In the pharmaceutical industry, antibiotic production now relies not on the original microbial strains, but on more productive mutants. The fungal strain discovered by Fleming synthesized only about 3 µg of penicillin per 1 ml of medium. Modern producer strains yield at least 2000 times more. Such an increase in antibiotic yield is the result of mutation and selection of more active strains, improvements in nutrient media composition, and optimization of production conditions. The Biosynthesis pathways of many antibiotics have already been elucidated, and the goal now is to further enhance microbial productivity through the generation and more targeted selection of mutants.

10.4.2 Mycotoxins

Mycotoxins are secondary metabolites produced by certain species of fungi. In a broad sense, fungal antibiotics can also be classified among them. In a narrow sense, however, mycotoxins refer exclusively to those fungal metabolic products that are toxic to higher animals and humans. A known producer of mycotoxins, for instance, is the aforementioned ergot fungus, Claviceps purpurea. Mycotoxins recently recaptured intense attention after thousands of turkey poults died from feed contaminated with aflatoxins. Aflatoxins (coumarin derivatives) are synthesized by individual strains of Aspergillus flavus, A. parasiticus, A. oryzae, and several other fungal species. They may occur in various moldy foods and feeds (peanuts, grain, oilseed fruits, animal feed). Aflatoxins possess carcinogenic properties.

Mycotoxins also include toxins from poisonous Basidiomycetes such as Amanita phalloides (amanitatoxin), A. pantherina, A. muscaria, and Inocybe patouillardii (fungal atropine and muscarine).

10.4.3 Other Secondary Metabolites

Among the Vitamins produced by microorganisms, riboflavin and vitamin B₁₂ deserve mention. Riboflavin is produced primarily by ascomycetes (Ashbya gossypii and Eremothecium ashbyii); however, yeasts (Candida) and bacteria (Clostridium) also synthesize flavins in large quantities. The ability to produce vitamin B₁₂ is characteristic of bacteria in whose metabolism corrinoids play a major role (Propionibacterium, Clostridium). This same vitamin is also produced by streptomycetes. Carotenoids used as animal feed additives are obtained from the mycelium of zygomycetes (Blakesleea trispora and Choanephora circinans).

As for Alkaloids, ergot alkaloids are the sole representatives obtained from microorganisms. Although ergot sclerotia (Secale cornutum) are currently harvested mainly by artificially infecting rye with the ergot fungus (Claviceps purpurea), submerged culture of certain Claviceps paspali strains is also gaining economic interest. Ergot alkaloids, which are lysergic acid derivatives (ergotamine, ergotoxine), are used in the Treatment of Vascular Diseases and migraines, and are also known as hallucinogens.

The principles of cultivating bacteria, yeasts, and other fungi are increasingly being applied to the cultivation of animal and plant cells as well. Methods have been developed for growing plant cells on synthetic media in bioreactors with capacities of thousands of liters. Under such conditions, plant cells produce enzymes and secondary metabolites at concentrations that can be 1–2 orders of magnitude higher than in intact cells. Surprisingly, such cultures may also accumulate substances that the plant synthesizes only in small quantities or does not synthesize at all. Eventually, it will likely become possible to produce alkaloids, Glycosides, Steroids, organic acids, and other secondary metabolites using plant cell cultures.

10.4.4 Other Microbial Products

Microorganisms can be used to produce a variety of Polysaccharides, enzymes, Antigens, and toxins. These microbial products Complement substances obtained by classical methods and, in some cases, replace them entirely.

Plant mucilages have long been used to increase liquid viscosity. Nowadays, they are increasingly being displaced by numerous bacterial exopolysaccharides (Section 2.2.4) (Table 10.1). Alginates are used as additives in ice cream, puddings, and creams. They have also found application as hydrophilic coatings to keep plant roots moist. Polysaccharides extracted from seaweeds are gradually being replaced by similar products obtained from Azotobacter or Pseudomonas. Xanthan Gums—mucilages produced by the phytopathogenic bacterium Xanthomonas campestris—have found versatile applications. Their backbone consists of glucose chains linked by ß-1,4-glycosidic bonds (just like in Cellulose) and bearing trisaccharide side chains. Xanthans are used as fillers in the food and cosmetic industries, as emulsion stabilizers for printing inks, and even as additives in oil-field drilling muds. Curdlans, which are not digested in the human intestine, are used to prepare puddings and low-calorie soups. Section 2.2.4 already discussed The Use of dextran as a Blood Plasma substitute and as a matrix for adsorbents known commercially as Sephadex.

Table 10.1. Exopolysaccharides synthesized by microorganisms and their applications

¹ A mixture of olive oil, vinegar, and mustard — Trans.

Enzyme preparations derived from animals and plants are also being gradually replaced by microbial enzymes (Table 10.2). Alongside Pepsin, Trypsin, and Papain, proteases secreted by aerobic spore-forming bacteria and fungi are widely employed. Instead of rennet, previously extracted from calf stomachs for milk coagulation, the enzyme rennin—produced by the fungus Mucor rouxii and certain other fungi—is now used.

Table 10.2. Enzymes synthesized by microorganisms and their applications

Sprouted grain is no longer used to convert starch into sugar for alcohol production; instead, fungal amylases are added to the starch. Invert sugar, used in confectionery, is produced from sucrose using invertase derived from Aspergillus oryzae and other fungi. Fructose, required for the manufacture of artificial honey and syrups, is obtained by converting glucose using hexose isomerase, sourced from certain species of Streptomyces or Lactobacillus brevis. Ongoing research is exploring ways to utilize inexpensive cellulose derived from wood and straw for ethanol production via microbial cellulases (sourced from Trichoderma viride, Penicillium). Recently, METHODS FOR PRODUCING many Other Enzymes have also been undergoing intensive development; to enhance their stability and handling, these enzymes are used in an immobilized form—that is, bound to a carrier material (such as cellulose, agarose, or glass beads).

For a more in-depth Study of the topics presented here, textbooks on industrial microbiology or biotechnology can be recommended. Introduction/32.html">Genetic Engineering (molecular cloning techniques, Section 15.3.6) has opened up new possibilities for biotechnology in this field as well.