Bioactive Products from Streptomyces

Adv. Appl. Microbiol. 47, 113-156, 2000. Bioactive Products from Streptomyces

VLADISLAV BĚHAL Institute of Microbiology Academy of Sciences of the Czech Republic Prague, Czech Republic

I. Introduction II. Chemistry and biosynthesis A. Peptide and peptide-derivative antibiotics B. Polyketide derivatives C. Other groups of bioactive products III. Genetics and molecular genetics A. Preparation of high production microorganisms B. Genetic manipulation of secondary metabolites producers IV. Obtaining new bioactive secondary metabolites A. Isolation from natural resources B B. Producers of bioactive compounds C C. Screening D. Semisynthetic and synthetic bioactive products E. Hybrid bioactive products and combinatorion biosynthesis V. Regulation of secondary metabolites production A.Growth phases of microbial culture B. Control of fermentation by basal nutrients C. How signals from the medium are received D. Regulation by low molecular compounds E. Autoregulators F. Regulation by metal ions VI. Resistance to secondary metabolites A. Resistance of bioactive secondary metabolites producers B. Resistance in pathogenic microorganisms VII. References

I. Introduction

A. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS

Medicine of twentieth century, especially its second half, was transformed by the discovery of antibiotics and other bioactive secondary metabolites produced by 2

microorganisms. Antibiotics are defined as microbial products that inhibit the growth of other microorganisms. After the antibacterial effect of penicillin had been observed by Fleming, a number of other antibiotics were discovered, mainly those produced by soil Streptomyces and moulds. Moreover, a broad spectrum of natural products having other effects on living organisms were found in microorganisms. In addition to standard antibiotics, the following compounds have also been found: coccidiostatics used in poultry farming, antiparasitic compounds with a broad spectrum of activity against nematodes and arthropods, substances with antitumor activity, immunosuppressors, thrombolytics (staphylokinase), compounds affecting blood pressure, end so forth. Microbial metabolites also exhibit good herbicide and pesticide activities and are biodegradable. However, microbial herbicides and pesticides only exceptionally used (e.g. bialaphos) due to their high price. Another special group of natural products are the enzyme inhibitors synthesized by microorganisms (Umezawa et al., 1976). These compounds can inhibit antibiotic derading enzymes, as well as certain enzyme activities in human metabolism that cause illness. Many enzyme inhibitors are protease inhibitors, variously active against pepsin, papain, trypsin, chymotrypsin, catepsin, elastase, renin, etc. Inhibitors of glucosidases, cyclic AMP phosphodiesterase, different carbohydrases, esterases, kinases, phosphatases, etc. have been also isolated from Streptomyces. The enzyme inhibitors that block synthesis of cholesterol are also important. Other exhibit the immunosuppressive effects, the most famous of them being cyclosporin A (a cyclic undecapeptide) produced by filamentous fungi. Some macrolide antibiotics, isolated from Streptomyces, are also immunosuppressives. Several thousands biologically active compounds have been deseribed and each year new compounds are isolated from microorganisms. Microorganisms are a virtually unlimited source of novel chemical structures with many potential therapeutic applications. The therm "secondary metabolite" used for some microbial products Bu´Lock (1961) and suitability of this therm discused Bennett and Bentley (1989). Secondary metabolites are meant compounds that the microorganism can synthesize but they are not essential for basic metabolic processes such as growth and reproduction. Nevertheless many secondary compounds function as the so-called signal molecules, used to control the producer’s metabolism. Another function attributed to antibiotics is a suppression of competing microorganisms in the environment whereby the antibiotic- producing microorganisms have an advantage in competing for nutrients with the other microorganisms. The production of secondary metabolites in microorganisms isolated from nature is rather low in most cases.To be usable for the commercial production of secondary metabolites, high yilding strains need to be selected through multiple mutations of the strain´s genetic material, optimization of culture conditions and genetic engineering.

II. Chemistry and biosynthesis

In spite of variety of their structures, bioactive secondary metabolites are synthesized from simple building units used in living organisms for the biosynthesis of cellular structures. These units include amino acids, acetate, propionate, sugars, nucleotides, etc.

According to their structure and type of biosynthesis, secondary metabolites are classified to form several groups.

A. PEPTIDE AND PEPTIDE-DERIVATIVE ANTIBIOTICS

Microorganisms produce a number of peptides as secondary metabolites. These peptide antibiotics are not synthetized on ribosomes but on enzyme complexes called peptide synthetases (Lipmann et al., 1971; Laland and Zimmer, 1973). In peptide antibiotic the peptide chain is often cyclic or branched. In addition to L-amino acids, other compounds can also be present in the molecule, such as D-amino acids, organic acids, pyrimidines and sugar molecules. The wellknown bioactive peptides, gramicidins and bacitracins are produced by different strains of Bacillus licheniformis and Bacillus brevis but some of them are produced by Streptomyces (Kleinkauf and von Doehren, 1986). The linear molecule of gramicidin A (Fig. 1) and the cyclic molecule of gramicidin S (Fig. 2) belong to the structurally simplest class of peptide antibiotics. Bacitracins are an example of cyclic peptides having a side chain (Fig. 3). In the molecule of bleomycin, the sugars L-glucose and 3-O-carbamoyl-D-mannose are found. Peptide antibiotics containing an atom of iron or phosphorus in the molecule have also been isolated. If two molecules of cysteine are present in the peptide antibiotic, they are linked by a sulfide bridge. The -CO-O- bond in the antibiotic molecule is present in lactones. Such antibiotics are represented especially by the group of actinomycins that contain a phenoxazine dicarboxylic group bearing two peptide chains. The enniatine molecule consists of three residues of branched amino acids, L-valine, L-leucine and L-isoleucine, and three residues of D-2-hydroxyisovaleric acid (D-Hyiv) (Billich and Zocher, 1987). The amino acids and D-Hyiv are linked by alternating amide and ester bonds. The amide bonds are finally N-methylated. Molecular conformation is important for the biological activity of peptide antibiotics. especially for the peptides capable of formating of chelates with metals. Studies showed three-dimensional molecular structures with many hydrogen bonds (Iitaka, 1978). In the + + case of valinomycin (L-Val-D-Hyiv-D-Val-L-Lac)3, which transports K and Rb ions across natural and artificial membranes, the molecule is symmetrical in three dimensions if it forms a complex with the metal. If it is not in the form of the complex, it has only a pseudocentral symmetry. The biosynthesis of peptide antibiotics takes place on a multienzyme complex. Kleinkauf and von Doehren,1983; Kleinkauf and von Doehren, 1986) The individual amino acids are activated using ATP to form aminoacyl adenylates. The aminoacyl groups are transferred to the enzyme thiol groups where they are bound as thioesters. The structural arrangement of the thiol groups in the synthetases determines the order of amino acids in the peptide. The formation of peptide bonds is mediated by 4- phosphopantetheine, an integral part of the multifunctional multienzyme. The intermediate peptides are also bound to the synthetases by the thioester bond. The way in which the order of the amino acids in the molecule is regulated is not known. It is probably determined by the tertiary configuration of the enzyme.

Our knowledge of the biosynthesis of peptide antibiotics comes mostly from the study of the gramicidin S and bacitracin synthetases. Gramicidin S synthetase consists of two complementary enzymes having molecular weights of 100 kD and 280 kD while bacitracin synthetase consists of three subunits (Roland et al., 1977) (Fig. 4) having molecular weights of 200, 210 and 360 kD (Ishiara et al., 1975). Each subunit contains phosphopantetheine. Enzyme A activates the first five amino acids of bacitracin, enzyme B activates L-Lys and L-Orn, and the enzyme C activates the other five amino acids. D-amino acids are produced by racemization of their L-forms directly on enzyme complex. Initiation and elongation start on subunit A up to the pentapeptide, independently of the presence of the subunits B and C. The pentapeptide is transferred to subunit B where two other amino acids are added. The heptapeptide is subsequently transferred to subunit C where the biosynthesis of bacitracin is finished. The cyclization is achieved by binding the asparagine carboxy group to the epsilon-amino group of lysine, whereas, the isoleucine carboxyl group is bound to the alpha-amino group of the same lysine (Laland et al., 1978). The antibiotic activity of bacitracin results in an efficient inhibition of proteosynthesis and cell wall synthesis but other effects such as an interference with cytoplasmic membrane components and cation-dependent antifungal effects have been observed as well. In the case of gramicidin S, hemolytic effects, inhibition of protein phosphatases and interaction with nucleotides have been observed in addition to the antibacterial activity. Even though antibiotics normally have several mechanisms of action, the primary one is defined to be the effect observed at the lowest active concentration. The peptide antibiotics are efficient mainly against Gram-positive bacteria. The b-lactams are peptide derived secondary metabolites. They are produced by different microorganisms . Several review sumarise reseach in these area (Martin and Liras, 1989; Jensen and Demain, 1995). The main producers are fungi (penicillins) but they are produced also by Strepromyces ( clavulanic acid) and Cephalosporium (cephalosporins). The main representatives of ß-lactams are penicillins and cephalosporins. Penicillins have a thiazoline ß-lactam ring in the molecule and differ, one from another, by side chains linked via the amino group (Fig. 5). Cephalosporins have a basic structure similar to that of penicillins and the derivatives are also formed by a variation of the side chain. The thiazolidine ß-lactam ring is synthesized using three amino acids: L-alpha-amino adipic acid, L-cystein and L-valine. By condensation of these three amino acids, a tripeptide is formed. It is transformed to the molecule of penicillin or cephalosporin through subsequent transformations (Fig. 6). Clavulanic acid, produced by Streptomyces clavuligerus, also belongs to ß- lactamfamily (Reading and Cole, 1977). This acid has a bicyclic ring structure resembling that of penicillin, except that oxygen replaces sulfur in the five-membered ring (Fig. 7.). Clavulanic acid is an irreversible inhibitor of many ß-lactamases. The discovery of clavulanic acid was a starting point for the development of penicillin analogues able to inactivate these enzymes. Penicillins are especially active against Gram-positive bacteria but some semisynthetic penicillins, such as ampicillin, that is lipophilic as compared to, for example, benzyl penicillin, are also effective against Gram-negative bacteria. This

effect is explained by their easier entering the cells of Gram-negative bacteria that have a high lipid content in the cell wall. ß-lactam antibiotics interfere with the synthesis of bacterial cell wall and thus inhibit bacterial growth. Such a mechanism of action does little harm to the macroorganism to which ß-lactams are applied. Another example of amino acid bioactive substances are the glycopeptides including semisynthetic derivatives (Zmijewski Jr. and Fayreman, 1995). The best known of all is vancomycin (Fig. 8) (Harris and Harris, 1982), effective against gram-positive bacteria. This antibiotic is widely used in medicine, especially against ß-lactam resistant strains. Vancomycin is not absorbed from the gastrointestinal tract and is used to treat enterocolitis caused mainly by Clostridium difficile. Vancomycin is produced by many species, of which Amycolotopsis orientalis is used for commercial production. Glycopeptides are composed of either seven modified or unusual aromatic amino acids or a mix of aromatic and aliphatic amino acids. By the substitution of amino acids in the amino acid core, derivatives of amino glycosides are formed. In vancomycin the aminosugar vancosamine is bound to the amino acid core. The removal of aminosugar reduces the activity of vancomycin two- to fivefold. The sugars seem to play an important role in imparting the enhanced pharmacokinetic properties for vancomycin-type, glycopeptide antibiotics.

B. POLYKETIDE-DERIVATIVES

Polyketides are a large group of secondary metabolites synthesized by decarboxylative condensation malonyl units often with subsequent cyclization of the polyketo chain . The starter group may be an acetate but also pyruvate, butyrate, ethyl malonate, paraaminobenzoic acid, etc. The formation of the initial polyketo chain is similar to that taking place during the biosynthesis of fatty acids, and is catalyzed by polyketide synthases. Simple carboxylic acids are activated as thioesters (acyl-SCoA) which are carboxylated to form malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA and after decarboxylation polymerized. ( Lynen, and Reichert, 1951; Lynen, 1959; Lynen and Tada, 1961). A principal role is played by the Acyl Carrier Protein (ACP) (Goldman and Vagelos, 1962). ACP detected throughout the growth of Streptomyces glaucescens was purified to homogenity and found to behave like many othes ACPs from bacteria and plants (Sumers et al. 1995). The ACP prosthetic group in many microorganisms is 4´-phosphopantothenic acid. Its terminal groups and acyls produced by polymerization are bound via the -SH group. The acyls are transferred to the other -SH group, that is a part of the cysteine molecule. Polyketide synthases have not yet been isolated and their properties have been deduced from the analyses of DNA sequences of cloned genes. Polyketide synthases include two distinct groups located either in domains on multifunctional proteins or present on individual, monofunctional proteins (McDaniel et al., 1993, Shen and Hutchinson, 1993). The structure and function of polyketide synthase in antibiotics overwie Robinson (1991) and Bentley and Bennett (1999). 6-Methyl salicylic acid (6MS) represents one of the simplest polyketides formed by condensation and subsequent aromatisation of one acetylCoA molecule and three malonylCoA molecules. This compound was isolated from Penicillium patulum (Bu ´Lock and Ryan, 1958). By other metabolic steps 6MS is transformed to produce a toxin

called patulin (Sekiguchi, 1983; Sekiguchi et al., 1983). The synthesis of 6MS takes place on an enzymatic complex called 6MS synthetase (Fig. 9) (Dimroth et al., 1970,1976). The chemical structure of sometypical tetracyclines is shown in Fig. 10 and their biosynthesis in Figs. 11 and 12 (McCormick, 1965). Chlortetracycline (CTC) and tetracycline (TC) are produced by the actinomycete Streptomyces aureofaciens, whereas oxytetracycline (OTC) and tetracycline by the actinomycete Streptomyces rimosus. For a more extensive coverage of research, articles by Běhal et al. (1983), Běhal (1987) and Běhal and Hunter (1995) should be consulted.

Tetracyclines act as inhibitors of proteosynthesis. They are considered to be wide- spectrum antibiotics that are efficient against both Gram-positive and Gram-negative bacteria. However, having significant side effects on the human macroorganism, they are preferably used only in the case where other, less toxic antibiotics are not effective. Anthracyclines are synthesized in a similar way as tetracyclines, however, they often have one or several sugar residues in the molecule. Most often deoxy-sugars, synthesized from glucose, are present in the anthracycline molecule. Daunorubicin and doxorubicin (adriamycin) (Fig. 13) are excellent antitumor agents, which are widely used in the treatment of a number of solid tumors and leukemias in human. Unfortunately, these drugs have dose limiting toxicities such as cardiac damage and bone marrow inhibition. In recent years, a variety of drug delivery systems for anthracyclines have been reported. In most cases, the drugs were linked to high molecular compounds such as dextran (Levi-Schaff et al., 1982; Tanaka, 1994), DNA (Campeneere, 1979), and others. Anthracyclines are produced by many Streptomyces (Grein, 1987) and genetics of their production is well elaborated (Hutchinson, 1995). Macrolides are usually classified to include: proper macrolides having 12-, 14- or 16-membered macrocyclic lactone ring to which at least one sugar is bound, and polyenes having 26- to 38-atom lactone ring containing 2 to 7 unsaturated bonds. Besides the sugars bound to the lactone ring, an additional aromatic part is normally present in the polyene molecule. Both macrolides and polyenes are biosynthesized in the same way using identical building units. Macrolides represent a broad group of compounds and new substances have been incessantly added to the list. Macrolides usually possess an antibacterial activity whereas polyens are mostly fungicides. Erythromycins produced by Saccharopolyspora erythrea (Fig. 14), together with oleandomycin and picromycin, belong to the best known 14-membered lactone ring macrolides (Harris et al., 1965). Macrolides with a 16-membered ring are represented by tylosin (Fig. 15) (Omura et al., 1975), that is produced by Streptomyces fradiae , as well as by leucomycin, spiramycin, etc. The synthesis of lactone ring is similar to that observed in the case of other polyketides. In contrast to aromatics, pyruvate and butyrate units are more often used in the biosynthesis, instead of acetate ones. The greatest difference, however, consists in the fact that, instead of aromatic rings, a lactone ring is formed. Keto- and methyl groups of the polyketide chain, from which macrolides are formed, are normally transformed more frequently. Nystatin is the best known polyene secondary metabolite (Fig. 16). Candicidine is another well known secondary metabolite belonging to the polyene group. Its molecule

includes p-aminoacetophenone as the terminal group. 4-amino benzoic acid (PABA) was identified as a precursor of the aromatic part of candicidine molecule (Liu et al., 1972, Martin, 1977). The sugars found in macrolide and polyene molecules are not usuallyencountered in microbial cells. They include both basic and neutral sugar molecules and L-forms are often found. So far, at least 15 different sugars have been described to occur in macrolides and polyenes. All of them are 6-deoxy sugars; some of them are N- methylated, others have the methyl on either the oxygen or carbon atom. As it has been repeatedly proven (Corcoran and Chick, 1966), glucose is primarily incorporated into macrolide sugar residues. Also in Streptomyces griseus, glucose, mannose and galactose were incorporated to a greater extent into the mycosamine candicidine, as compared to its aglycon (Martin and Gil, 1979). The transformation of glucose to a corresponding sugar takes place in the form of the nucleoside diphosphate derivatives, which is similar to the situation found in the case of other secondary metabolites. Avermectins consist of a 16-membered, macrocyclic lactone to which the disaccharide oleandrose is bound (Fig. 17) (Burg, R.W., 1979; Miller, T.W., 1979). Avermectins are produced by Streptomyces avermitillis. The macrocyclic ring of avermectins is synthesized, as other polyketides, by producing a chain from acetate, propionate and butyrate building units. Oleandrose (2,6-dideoxy-3-O-methylated hexose) is synthesized from glucose. Avermectins are potent antiparasitic compounds active against a broad spectrum nematode and anthropod parasites. They lack antifungal and antibacterial activities. They bind to a specific, high-affinity site present in nematodes but not in vertebrates. Its dosage for animal and human is extremely low. Ivermectin (22,23-dihydroavermectin B1) is a semisynthetic compound which is used to control internal and external parasites in animals and is the most potent anthelmintic compound of all. Avermectins are also employed in human medicine and plant protection. Detailed reviews on the uses and biosynthesis of avermectins can be found in recent monographs (MacNeil, 1995; Ikeda and Omura, 1995). Polyethers form a large group of structural related natural products mainly produced by Streptomyces (Birch and Robinson, 1995). They are potent coccidiostats (monensin, salinomycin) and are used in the agricultural arena.(Westley, 1977). Polyethers are compouns possesing the ability to form lipid-soluble complexes that provide a vehicle for a wide variety of cations to traverse lipid barrieres. This ion- bearing property led to their being named ionophores (Moore and Pressman, 1994). Backbones of polyethers are synthetized from acetate, propionate and butyrate (monensin A) units. Isobutyrate and n-butyrate are efficiently incorporated into polyether antibiotics (Pospíšil et al., 1983). Incorporation of isobutyrate was explained by formal conversion of isobutyryl-CoA into n-butyryl-CoA or methylmalonyl-CoA by isobutyryl-CoA mutase and methylmalonyl-CoA mutase, respectively.

C. OTHER GROUPS OF BIOACTIVE PRODUCTS

Chloramphenicol (Fig. 18) is produced by Streptomyces venezuelae (Vining and Westlake, 1984). At present, however, the antibiotic is commercially produced using a

fully synthetic process. In contrast to polyketides, the aromatic ring of chloramphenicol molecule is synthesized from glucose via chorismic acid and p-amino benzoic acid in the microbe. Streptomycin (Fig. 19) is a well-known aminoglycoside antibioticoriginaly discovered by Selnon Waksman. It is synthesized by many streptomycetes to produce a number of derivatives. The molecule of streptomycin consists of three components: streptidine, L-streptose and N-methyl-L-glucosamine. None of these components has been found in the primary metabolism of microorganisms. The steps of streptomycin biosynthesis were disclosed mainly by Walker (Walker and Walker, 1971), who also studied the relevant enzymes (Walker, 1975). The importance of streptomycin consists mainly in its ability to suppress Mycobacterium tuberculosis, resulting in effective suppression of tuberculosis, especially in developed countries. Bialaphos is formed from two L-alanine residues and the amino acid phosphinothricine. The latter compound is synthesized by streptomycetes from acetylCoA and phosphoenolpyruvate, and subsequently methylated using methionine as the methyl donor (Bayer et al., 1972; Ogawa et al., 1973). The producing microorganisms are Streptomyces hygroscopicus and Streptomyces viridochromogenes. Bialaphos, as well as phosphinothricine, inhibits the activity of glutamine synthetase.

III. Genetics and molecular genetics

A. PREPARATION OF HIGH PRODUCTION MICROORGANISMS

The structural genes encoding the enzymes that synthesize secondary metabolites are mostly located on chromosomes They are often organized in gene clusters (Binnie et al., 1989; Malpartida and Hopwood, 1984; Lotvin et al., 1992; Martin, 1992). Resistance of the producer to its own products are located either at the beginning or at the end of the cluster, often in both positions. In addition to the resistance and structural genes, regulatory genes are important in secondary metabolites production, however, they function is poorly understand. Microorganisms that are isolated from nature (wild type strains) produce small amounts of secondary metabolites. Sometimes during selection and subsequent cultivation in the laboratory, a changes occur, making the cultivated strain non-identical with the original strain. In such cases it should be remarked that the term wild type strain only refers to the fact that the strain did not undergo an “artificial” genetic change. In order for the commercial production of secondary metabolites could be profitable, higher levels of the secondary metabolites synthesis are reached via genetic changes of producers. Mutants are isolated by exposure of spores to UV irradiation, X-rays, γ-rays, α-particles or chemical mutagens (nitrogen mustards, N-methyl-N´-nitro-N-nitroso guanidine). Combined mutagenesis using various mutagens is often used. The surviving spores give rise to individual colonies of isolates, whose capability of secondary metabolites production is then tested. Mutants that exhibit poor growth and sporulation ability are not suitable candidates for further improvement, even if their secondary

metabolites production may exceed that of the original strain. Today’s high production strains, that synthesize as high as 10 000-fold levels of secondary metabolites, compared to the original strains, are the result of many year of costly strain improvement. Unfortunately, these high production strains can revert to lose their overproduction though spontaneous mutagenesis. When high production strains are prepared by mutagenesis, a type of mutant that loses some of the structural genes can also be obtained. Such a mutant can exhibit a higher level of a secondary metabolite intermediate whose transformation stopped due to the absence of the corresponding enzyme. By crossing these mutants, some biosynthetic pathways used to synthesize secondary metabolites were elucidated, e.g. tetracyclines (McCormick et all.1960). Loss of the capability of secondary metabolite production in the strains where extrachromosomal DNA was removed (e.g. by using acriflavine or ethidium bromide) suggests that the regulatory genes are located on plasmids (Hotta et al, 1977; Okanishi, 1979; Akagava et al., 1979; Boronin et al., 1974; Ikeda et al., 1982).

B. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

Structural genes for a number of secondary metabolites have been cloned into host microorganisms. Similarly, genes for secondary metabolites resistance and other regulatory genes have also been cloned. Streptomyces lividans was found to be a suitable acceptor of foreign genetic material, in which a low degree of restriction of this genetic material exists. This microorganism can host various plasmids and phage vectors. However, at the same time, this microorganism was found not to be usable for the synthesis of various secondary metabolites or of their high levels. The secondary metabolites biosynthesis is a very complex process that requires not only the structural genes for ESM but also the genes for regulation of their biosynthesis. Moreover, the overproduction of a secondary metabolite has to be coordinated with the primary metabolism of the producing microorganism. The cloning of structural genes and genes for resistance to the own secondary metabolite enables us to work out genetic maps of the producers. On the basis of those maps, hybrid clusters combined of two and more clusters of different secondary metabolites can be created. Consequently, semisynthetic secondary metabolites can be produced that may possess new biological activities or an antibiotic activity against resistant strains. Polyketide synthase genes of microorganisms producing various polyketides have also been hybridized (Hopwood and Sherman, 1990). As a result, a great similarity of polyketide synthases from various streptomycetes was evidenced (Malpartida et al., 1987; Butler et al., 1990).

IV. Obtaining new bioactive secondary metabolites

A. ISOLATION FROM NATURAL RESOURCES

In spite of the fact that several thousands of compounds isolated from microorganisms having some biological activity are known, new substances are still saught by pharmaceutical companies. The probability of finding a new compound that would be usable as a new antibiotic or another biologically active compound is low, so a great number of microorganisms have to be screened. A rough estimation says that about 100 000 microorganisms are screened for the presence of biologically active compounds per year. Modern screens are highly automated. The selection methods used, the targets, and the methods of detection of the biological activity are normally not published. Preparation of a new biologically active compound and its introduction into clinical practice requires the cooperation of scientists from various scientific disciplines and years of clinical trials. This effort can be divided into three parts:(Yarbrough et al., 1993): 1. microbiology -collection of source samples (soil) -isolation of diverse microbes -fermentation to enhance diversity -reproduce fermentation -enhance the production for isolation -taxonomy of the organism 2. molecular biology/pharmacology -target selection -screen design/implementation -high through-put screening -identification of active compounds -efficacy studies -mechanism of action 3. chemistry -active compound identification -characterisation/dereplication -isolation/purification -structure elucidation.

B. PRODUCERS OF BIOACTIVE COMPOUNDS

About 70 % of the known bioactive substances are produced by Streptomyces and the rest mainly by moulds and non-filamentous bacteria. With an increasing spectrum of efficiency of microbial metabolites, new, non-traditional sources of such compounds have been tappede. These include the microorganisms living under extreme conditions (high and low temperatures, etc.), sea living microorganisms, and multicelular plants and animals. Another important source of new compounds are the mutants of producers of known active substances, e.g. blocked mutants.

B. SCREENING

The enterprise of screening microbial metabolites for new leads, first exploited by antibiotic researchers and today expanded to virtually all fields of therapeutic interest, has proven successful and will continue as an important avenue to new drug discovery. The original method for determination of antibiotic efficiency consisted of the application of test extract to wells made in agar medium layer in Petri dishes to which the sensitive (target) microorganism was inoculated. Most often Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella gallinarium, Pseudomonas spp., Bacillus subtilis, and Candida albicans were used. In case a compound with an antibiotic activity towards the testing microorganism was put into the well, it diffused through the agar medium and a halo was formed around the well, as a result of the suppressed growth of the microorganism. This classic plate assay has been modified and improved in many ways. The tests of other biological activities require different and frequently sophisticated methods. This is true especially when enzyme inhibitors are a case in point. Thus, Ogawara et al. (1986) chose a tyrosine protein kinase associated with the malignant transformation of the cell caused by retroviruses as the target in a biochemical screen, they found genistein, an isoflavone from Pseudomonas, exhibiting a specific inhibitory activity. Production of target enzymes using recombinant DNA methodology has dramatically expanded the number of potential targets that can be feasibly screened. A screen for the inhibitors of HIV reverse transcriptase is an example. The enzyme was produced in Escherichia coli, purified by affinity chromatography, and used to test natural products for the activity (Take et al., 1989).

D. SEMISYNTHETIC AND SYNTHETIC BIOACTIVE PRODUCTS

Natural products can be modified in various ways. The unspecificity of the enzyme systems facilitates the synthesis of certain secondary metabolites though the addition of selectedprecursors to the growth medium. Thus, the reaction equilibrium can be shifted to promote the production of the derivative required, e.g. the prepareation of penicillins with different side chains. The individual derivatives of penicillin and cephalosporin have slightly different antimicrobial spectra and are active against microorganisms resistant to other derivatives. The structuure of polypeptide antibiotics can also be modified by the addition of amino acids to the growth Replacement of a part of the metabolite molecule can be accomplished chemically or enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines and other antibiotics can be prepared. The production of semisynthetic penicillins and cephalosporins is facilitated by the fact that 6-amino penicillanic and 7-amino cephalosporanic acids are easily prepared. The side chain is removed by the action of an enzyme or by a chemical hydrolysis (Fig. 20) then another acyl is bound chemically or enzymatically to the amino group in position 6 (penicillins) or 7 (cephalosporins). Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline, exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared to the original tetracyclines.

New derivatives of aminoglycosides also have been obtained by chemical and enzymatic modifications. As the majority of bioactive products have rather complex structures, their chemical synthesis is mostly more expensive than the production by fermentation. An exception to the rule seems to be chloramphenicol, that is normally prepared using a chemical synthesis.

E. HYBRID BIOACTIVE PRODUCTS

Genetic engineering methods have recently advanced so much that now we can suitably combine structural genes of two or even more bioactive secondary metabolites producers. If these genes are expressed, a hybrid bioactive products is synthesized, ore that cannot be found in nature (Hutchinson, 1987, 1988; Tomich, 1988; Hopwood, 1993). Hopwood et al. (1985, 1986,) used this method with the genes of actinorhodin synthesis and obtained related hybrid macrolides, mederhodin A and B, dihydromederhodin A and dihydrogranatirhodin. A new anthracyclines were produced when a DNA segment was cloned from Streptomyces purpurascenc ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase Streptomyces galilaeus ATCC 31615 (Niemi et al., 1994).

V. Regulation of secondary metabolites production

A. GROWTH PHASES OF Stepromyces

In the cultures of Streptomyces capable of secondary metabolite production several growth phases representing different physiological statescan be distinguished:

1. Preparatory phase (lag phase) - the biomass increase is low, the culture is adapting to the new environment. 2. Growth phase (the term logarithmic phase is not suitable for most Streptomyces since their growth curves are not exponential functions) - intensive growth is taking place, accompanied by a low secondary metabolite synthesis. This phase is roughly equivalent to "trophophase". 3. Transition phase - characterized by a decreased growth rate; the secondary metabolite production is started. The enzymes of secondary metabolism are synthesized (Běhal, 1986a; Běhal, 1986b) and proteosynthesis slowed down. 4. Production phase - characterized by a significant reduction of the growth rate (sometimes growth is even completely ceased), a negligible change in the biomass concentration, and an intensive synthesis of the secondary metabolite. This phase is some times called "idiophase".

Producers of secondary metabolites mostly belong filamentous bacteria or fungi, which means that in their culture cells of various age and at different stages of

development are present. The microorganisms grow in pellets, inside which the cultivation conditions differ from those on the pellet surface (nutrient concentrations, oxygen concentration, etc.). An increase in dry weight does always correlation with an increase growth since, in streptomycetes, often a thickening of the cell wall or glycocalyx formation occur that increase the dry weight value without rising the number of cell (Voříšek et al., 1983). Since individual cells of a fermentation can be at different stages of development, (i.e. in different physiological states). The physiological state of the whole culture represents an average of physiological states of the individual cells.

B. CONTROL OF FERMENTATION BY BASAL NUTRIENTS

In order to reach a high yieldof secondary metabolite, sufficient biomass is required. Moreover to danger of contamination is diminished and the economic parameters of the fermentation device are optimal if the growth is rapid. For this purpose, readily utilizable sources of carbon, nitrogen and phosphorus sources (e.g. molasses, corn starch, etc) are used. However, production of the secondary metabolite does not usually take place until one or more nutrients become limited.Thefore, the culture medium should be designed in such a way that after the biomass increased sufficiently, at least one of the nutrient sources will become depleted. Carbon source, nitrogen source and phosphate limitation have been described as important triggers in different systems. Most secondary metabolites are produced in a fed batch system, i.e. a certain amount of the culture medium is inoculated with the producing microorganism and, after a time interval, another dose of nutrients is added to the fermenter. Thus a prolonged cultivation can be accomplished that enables us to increase the yield of the secondary metabolite. The inflow of nutrients makes possible keep their optimal levels. An example of how a production cultivation of Streptomyces aureofaciens can look like is shown in Fig. 21 (Běhal, 1987). In cultivations whose course is well known, the nutrient inflow is programmed in advance

The inhibition of penicillin synthesis by glucose was observed shortly after its discovery in media containing glucose and lactose (Demain, 1974). The antibiotic was found to be synthesized only after glucose was depleted from the medium and lactose started to be metabolized. Similarly, glycerin was observed to inhibit the biosynthesis of cephalosporins (Demain, 1983). Using these data, fermentation protocols were worked out, in which the level of glucose was kept low so as not to inhibit the antibiotic production. The mechanism of inhibition of the secondary metabolites synthesis by readily utilizable sugars probably consists in a repression of enzymes of secondary metabolism (Revilla, G. et al., 1986; Erban, et al., 1983). Readily utilizable nitrogen sources can also negatively influence the production of secondary metabolites.Ammonium ions often decrease secondary metabolite synthesis and, therefore, their concentration in production media is limited while, soy flour, peanut flour and other substances are preffered nitrogen sources. These latter nitrogen sources are more similar to those used by the microorganisms producing secondary metabolites in nature. Readily utilizable nitrogen sources repress enzymes of secondary metabolism in Cephalosporium acremonium (Shen et al., 1984) during the biosynthesis of cephalosporin and in Streptomyces clavuligerus producing cephamycin (Demain and

Brana, 1986). Similarly, the inhibition of biosyntheses of leucomycin (Omura et al., 1980a), tylosin (Omura et al., 1980b), and erythromycin (Flores and Sánches, 1985) are explained by the repression of enzymes of secondary metabolism. Ammonium salts also inhibit the activity of anhydrotetracycline oxygenase isolated from S. aureofaciens (Běhal et al., 1983). The overproduction of most secondary metabolites can be achieved only if phosphate is limited. Inorganic phosphate has to be carefully added in doses to the medium so as to accomplish an optimal ratio between biomass production and secondary metabolite biosynthesis. When bound to organic compounds normally added to medium (soy flour, etc.), phosphate does not affect secondary metabolite production. In general secondary metabolite biosynthesis is started when the concentration of phosphate decreased below a certain level. At this point, the producer culture undergoes a shift from the physiological state characteristic for the growth phase to that of the overproduction phase. Inorganic phosphate also causes a repression of the synthesis of enzymes of secondary metabolism (Běhal et al., 1979b; Madry and Pape; 1981, Martin et al., 1981). After phosphate was depleted from the medium, a significant decrease of the rate of proteosynthesis was observed during tetracycline (Běhal,1982). If phosphate was kept above the threshold concentration, the significant decrease of the rate of protein synthesis did not occur and ESM were not synthesized. An addition of phosphate to the medium at the beginning of the production phase, after the phosphorus source was depleted and the enzymes of secondary metabolism synthesis initiated, resulted in a decrease of the enzymes of secondary metabolism levels in the culture and an acceleration of proteosynthesis.

C. HOW SIGNALS FROM THE MEDIUM ARE RECEIVED

Reception of signals from the environment, that result in the initiation of the secondary metabolite synthesis does not significantly differ from the transduction of signals for other metabolic processes. Catabolite repression signals or those signalling the depletion of nitrogen or phosphate, or the initiation of sporulation, are transducted via two-component signal proteins ( Doull and Vining, 1995). With some structural varietion, these proteins are characterized by common mechanistic features and conserved amino acid sequences. The two-component system consists of a cytoplasmic membrane-linked, sensor- transmitter protein and a response-regulator protein, located in the cytoplasm. The sensor-transmitter is composed of a sensor domain located near its N-end; the N-end is found outside the cytoplasm. A specific effector is capable of binding directly to this N- end. The transmitter domain is located in the cytoplasm to be linked to the sensor domain via a hydrophobic, amino acid sequence stretching across the membrane. The sensor-transmitter proteins are histidine-protein kinases, capable of autophosphorylation at their C-ends on receiving a proper signal. The phosphorylated protein becomes a donor in reactions transferring phosphorus. The acceptor is the cytoplasmic, response-regulator protein. Two-component signal proteins thus transfer the information concerning the conditions that can affect the cell action.

D. REGULATION BY LOW MOLECULAR COMPOUNDS

The expression of structural genes is also regulated by some low molecular compounds. The mechanism of their action is not understood. For example tryptophan exhibited a stimulatory effect on the production of mucidin in the basidiomycete Oudemansiella mucida (Nerud et al., 1984) and actinomycin in Streptomyces parvulus (Troast et al., 1980). Methionine was found to promote the synthesis of cephalosporin C (Nuesch et al., 1973). Neither tryptophan nor methionine were used as building units for these metabolites. Benzyl thiocyanate is one of the low molecular compounds that affect the chlortetracycline biosynthesis. It increases the production of both chlortetracycline and tetracycline in S. aureofaciens, although, it does not influence the production of oxytetracycline in S. rimosus. The effect on the metabolism of S. aureofaciens is multiple (Novotná et al., 1995), including a number of enzymes, including the enzymes of secondary metabolism (Běhal et al., 1982). Benzyl thiocyanate is able to raise the level of secondary metabolite production only if it is added in the lag phase, growth phase or at the beginning of the production phase. Its effect is more pronounced in low production strains, where the enzyme level and chlortetracycline production are increased 10 to 20-fold, as compared to high production strains where the increase is only twofold. .

E. AUTOREGULATORS

Streptomycetes low-molecular, diffusible compounds have been discovered that regulate the metabolism of producing strain (Horinuchi and Beppu, 1990; Horinuchi and Beppu, 1992). The most famous of them is factor A, γ-butyrolactone (Fig. 22), that was discovered in Streptomyces griseus (Khokhlov et al., 1969; Khokhlov, 1982). A non-producing strain started the synthesis of streptomycin after factor A was added to the culture simultaneously, the coltura formed aerial mycelium. Factor A is synthesized by many streptomycetes but the regulatory effect was observed only in Streptomyces griseus, Streptomyces bikiniensis and Streptomyces actuosus (Ohkishi et al., 1988). The addition of factor A to blocked mutants of Streptomyces griseus JA 5142, caused resumption of the synthesis of anthracyclines and leukaemomycin (anthracycline type antibiotic) (Graefe et al., 1983). The resistance to streptomycin linked with an enzymatic phosphorylation of the antibiotic is also induced by factor A (Hara and Beppu, 1982). Analogues of factor A have also been found, all of them being γ-butyrolactones. Virginiae butanolides were detected in Streptomyces virginiae (Yanagimoto et al., 1979). Factor I was isolated from Streptomyces sp. FR1-5 (Sato et al., 1989) and its effective concentration was 0.6 ng/ml culture. Most of the factor A analogues, however, were not biologically active. Factor B was isolated from the yeast Saccharomyces cerevisiae. This substance was capable of eliciting the production of rifamycin in a blocked mutant of Nocardia sp. (Fig. 23) (Kawaguchi et al., 1984). Factor B was effective at a concentration of 10-8 M, with one molecule eliciting a synthesis of about 1500 molecules of the rifamycin. The

structure of factor B is similar to cAMP but none of the derivatives of known nucleotides exhibited a comparable effect. Chemically prepared derivatives of factor B have also been tested. Activity was observed with those that contained a C2 -C12 acyl moiety; octylester was the most effective of them (Kawaguchi et al., 1988). A substitution of guanosine for adenine did not result in a loss of the biological activity of factor B. Factor C was isolated from the fermentation medium of Streptomyces griseus. This compound causes cytodifferentiation of non-differentiating mutants (Szabo et al., 1967). Factor C is a protein having a molecular weight of about 34 500 D, and is rich in hydrophobic amino acids. The effect of autoregulators is easily observable if they elicit morphological changes such as the formation of aerial mycelium. Carbazomycinal and 6-methoxcarbazomycinal, isolated from Streptoverticillium species, inhibit of the aerial mycelium formation at a concentration of 0.5 to 1 microgram per ml. Autoregulators affecting sporulation were found in Streptomyces venezuelae (Scribner et al., 1973), Streptomyces avermitilis (Novák et al., 1992), and Streptomyces viridochromogenes NRRL B-1551 (Hirsch and Ensign, 1978). From the same strain of Streptomyces viridochromogenes, germicidin was isolated by Petersen and coworkers (1993). The compound had an inhibitory effect on the germination of arthrospores of Streptomyces viridochromogenes at a concentration as low as 40 picogram per ml. Germicidin (6-(2- butyl)-3-ethyl-4-hydroxy-2-pyrone) is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores and also from the supernatant of a submerged culture. Mutants of Streptomyces cinnamonensis resistant to high concentrations of butyrate and isobutyrate produce an anti-isobutyrate (AIB) factor that is excreted into the culture medium (Pospíšil, 1991). On plates, AIB factor efficiently counteracted toxic concentrations of isobutyrate, acetate, propionate, butyrate, 2-methylbutyrate, valerate, and isovalerate in Streptomyces cinnamonensis and other Streptomyces species.

F. REGULATION BY PHOSPHORYLATED NUCLEOTIDES

Global control mechanisms for secondary metabolites biosynthesis have been investigated. The energetic state of the cell is thought to be such a general control mechanism. The intracellular ATP level reflects the content of free energy in the cell. In some cases, the start of the secondary metabolite synthesis is linked with a decrease of the intracellular ATP level. Such a relationship was observed in Streptomyces aureofaciens and Streptomyces fradiae during the production of tetracycline (Janglová et al., 1969; Čurdová et al., 1976) and tylosin (Madry et al., 1979; Vu-Truong et al., 1980), respectively. Even though the regulatory role of ATP cannot be strictly excluded, the results seem to support a hypothesis that a higher ATP level is accompanies active primary metabolism. A slow down of growth and primary metabolism is accompanied by a decrease of the ATP level. The role of cAMP in the metabolism of secondary metabolites producers was also studied, especially in connection with glucose regulation. Hitherto, no indication has

been obtained suggesting a specific role of cAMP in the regulation of secondary metabolites production (Cortéz et al., 1986; Chatterjee and Vining, 1981).

G. REGULATION BY METAL IONS

Metal ions act as a part of enzyme active centers. The optimal concentrations of metal ions for cultivation of the secondary metabolites producing strains have usually been determined empirically. In complex media it is generally not necessary to add specific metal ions, however in defined media their presence is essential.

VI. Resistance to bioactive products

Resistance against bioactive products has been studied mainly in antibiotic producers. Antibiotic resistance is usually looked at from two angles: first, the emergence of drug rezsstant strain and second, "self resistance" of antibiotics producing strains. The ways in which these two types of resistance are achieved is often similar.

A. RESISTANCE OF SECONDARY METABOLITES PRODUCERS

Basic metabolic processes of wild type, secondary metabolite producing microorganisms are not inhibited if the secondary metabolires are synthesized at low concentrations. After strain improvement, strains with 100 to 1000-fold increases insecondary metabolite yields have been isolated. Genome changes of the improved strains include a number of deletions and amplifications in the chromosomal DNA, as well as changes in extrachromosomal DNA. Low production strains, whose resistance to the own product is low (i.e. higher concentrations of the product inhibit their growth), regulate the secondary metabolite production by inhibiting the enzyme activities that participate in the synthesis of the secondary metabolite. In high production strains, such controls are lost and the strains have to find a way how to survive in the presence of a high concentration of the antibiotic (Vining, 1979). The genes for self resistance are often located at the beginning of the cluster of structural genes. As a result, they are expressed simultaneously with the structural genes. The genes of newly gained resistances, however, are mostly located on plasmids. Some antibioticsfunction by hitting active centres of enzymes. However, if active centre is modified, the antibiotic cannot bind to it and then resistance comes into existence. It is not known whether a decreased ability to bind the secondary metabolites results from a posttranslational modification of the active centre or if resistant molecules of the enzyme are synthesized de novo. Clear evidence in support of the latter situation has sofar been brought. Many antibiotics inhibit protein synthesis, the target site being at the ribosome level. Often, the functions of Tu and G elongation factors are also impaired, together with reduced synthesis of guanosine penta- and tetraphosphates (Weiser et al., 1981). The antibiotic producers (mostly Streptomyces), as well as the bacteria against which the antibiotic is used, protect themselves by posttranscriptional modification of rRNA.

Adenine is methylated to obtain N6-dimethyladenine rRNA in the 23S subunit. Such modified ribosomes do not bind the antibiotic. In other cases, adenine is methylated to yield 2-O-methyladenosine (Cundliffe and Thompson, 1979; Mikulík et al.,1983; Thompson et al., 1982). However, methylation modified ribosomes can be sensitive to the effect of other antibiotics. The genes coding for methylases, that catalyze methylation of adenine in some Streptomycetes, were cloned into Streptomyces lividans and the ribosomes of the mutants prepared were resistant towards the corresponding antibiotics. The most important mechanism of resistance observed in the secondary metabolites producers seems to be export from the cell to the environment. In Streptomyces rimosus, an oxytetracycline producer, genes for the enzymes increasing the antibiotic transport rate precede the structural genes on the chromosome. Genes for the resistance consisting in the protection of ribosomes via the synthesis of an unidentified protein are located at the end of the structural gene cluster(Ohnuki et al., 1985). Producers bioactive secondary metabolites also have to solve the problem of a reverse flow of products into the cell. Some secondary metabolites are bind to the cell wall, others are complexed in the medium (tetracyclines in the presence of Ca2+ ions). Cytoplasmic membranes of resistant strains are often less sensitive to the effect of secondary metabolites. This kind of resistance is thought to be connected with the content of phospholipids in the cell. Secondary metabolite producers can use several types of resistance simultaneously. Tetracyclines, that strongly inhibit protein synthesis, interfere with the binding of the ternary complex of amino acyl-tRNA-EFTu-GTP to ribosomes (Gavrilova et al., 1976). The genes for resistance were cloned into Streptomyces griseus, sensitive to tetracyclines, using pOA15 as the vector plasmid (Ohnuki et al., 1985). After mapping the plasmids in resistant strains using restriction nucleases, two types of plasmids capable of transfer of different types of resistance were found. One type consisted in an increased ability of tetracycline transport to the medium, the other in an increased resistance of ribosomes to the effect of tetracyclines. These ribosomes bore a compound(s), bound to their surface, that could be removed by washing with 1 M NH4Cl solution. The ribosomes lost their resistance after the washing, which was demonstrated with both the ribosomes of Streptomyces griseus and those of the original strain of Streptomyces rimosus. The two types of resistance were both constitutive and inducible. The inhibiting concentrations of chlortetracycline in Streptomyces aureofaciens are higher in the production phase as compared to the growth phase (Běhal et al., 1979a). Thus, the resistance can be increased even during the fermentation process. Another way secondnary metabolite producers can avoid the effect of their products is to situate the distal enzymes of secondary metabolite biosynthetic pathway (synthases) outside the cell, most often in the periplasm. In Streptomyces aureofaciens, a higher proportion of the terminal enzyme of tetracycline synthase was found under high production conditions in periplasm, as compared to low production conditions (Erban et al.,1985).

B. RESISTANCE IN PATHOGENIC MICROORGANISMS

Shortly after antibiotics were introduced into clinical practice on a massive scale, strains of hitherto-sensitive microorganisms started to appear. These resistant strains required the use of much higher antibiotic concentrations or, were completely resistant to these antibiotics. The resistant strains originated from clones that survived the antibiotic treatment, especially if the treatment was terminated before all pathogenic microorganisms were killed or the antibiotic was applied at sublethal doses. There are several ways in which microorganisms can gain resistance (Ogawara, 1981). These include: 1. Creation of an alternative metabolic pathway producing a compound whose biosynthesis is blocked by the bioactive metabolite; 2. Production of a metabolite that can antagonize the inhibitory effects of the bioactive metabolite; 3. Increase of the amount of the enzyme inhibited by the secondary metabolite; 4. Decrease of the cell’s metabolic requirement for the reaction inhibited by the secondary metabolite; 5. Detoxification or inactivation of the bioactive metabolite; 6. Change of the target site; 7. Blocking of the transport of the bioactive metabolite into the cell. In most resistant microorganisms, the mechanisms of resistance mentioned in the items 5, 6 and 7 are encountered. Penicillins and cephalosporins are degraded using three ways: by the enzyme penicillin amidase that cleaves the amidic bond by which the side chain is bound to the β-lactam ring; by the enzyme acetyl esterase that hydrolyzes the acetyl group at C-3 on the dihydrazine ring of cephalosporins and by the enzyme β-lactamase that catalyzes hydrolysis of the β-lactam ring of penicillins and cephalosporins. Penicillin amidases are rarely used by microorganisms to build up resistance to β- lactam antibiotics, however these enzymes are often employed for the synthesis of semisynthetic antibiotics. Acetyl esterase is also not important from the point of view of antibiotic resistance. In most cases, β-lactams are inactivated by β-lactamase that destroys one of the important sites for their antibiotic activity; the damage is irreversible. Β-lactamases, however, are not only synthesized by microorganisms that came into contact with penicillins. Constitutive synthesis of these enzymes have been found in three quarters of all streptomyces strains, (Ogawara et al., 1978). One can suppose that the genes for the synthesis of β-lactamases were transferred horizontally. Recent studies indicate frequent and promiscuous gene transfer even between distantly related bacterial species. A possibility of direct transfer from a streptomycete to a pseudomonad, for example, may seem unlikely. However, it is not necessary to invoke direct exchanges. It is more reasonable to imagine that distant exchanges between distantly related organisms result from a cascade of transfer between related species (Davis, 1992). Another way of inactivating a bioactive metabolite molecule is N-acetylation of the amino group or O-phosphorylation of the hydroxyl. Bialaphos was found to be inactivated by acetylation. These substance itself is not toxic but, in the cell, phosphinothricine is liberated that inhibits glutamine synthetases, key enzymes of the inorganic nitrogen assimilation pathway.

VIII. References

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Fig. 1. Gramicidin A Fig. 2. Gramicidin S Fig. 3. Bacitracin Fig. 4. Bacitracin synthetase Fig. 5. Penicillins Fig. 6. Cephalosporins Fig. 7. Clavulanic acid Fig. 8. Vancomycin Fig. 9. 6-methyl salicylic acid synthetase Fig. 10. Tetracyclines Fig. 11. Tetracycline biosynthesis Fig. 12. Tetracycline biosynthesis Fig. 13. Anthracyclines Fig. 14. Erythromycins Fig. 15. Tylosin and Relomycin Fig. 16. Nystatins Fig. 17. Avermectins A - R5= OCH3; B - R5= OH; 1 - X= -CH=CH-; 2 = X= -CH2-CHOH-; a - R26= C2H5; b - R26= CH3 Fig. 18. Chloramphenicol Fig.19. Streptomycines Fig. 20. 6-aminopenicillanic acid and 7-aminocephalosporanic acid Fig. 21. Parameters of an industrial fermentation of S. aureofaciens 1 - chlortetracycline production (g/l); 2 - ATC-oxygenase (pkat/ mg proteins x 2); 3 - NH3-nitrogen (g/l x 0.1); sucrose (g/l x 10); 5 - pH; ammonium supplement were added at points A and B Fig. 22. Factor A

Fig. 23. Factor B

Adv. Appl. Microbiol. 47, 113-156, 2000. Bioactive Products from Streptomyces

VLADISLAV BĚHAL Institute of Microbiology Academy of Sciences of the Czech Republic Prague, Czech Republic

I. Introduction II. Chemistry and biosynthesis A. Peptide and peptide-derivative antibiotics B. Polyketide derivatives C. Other groups of bioactive products III. Genetics and molecular genetics B. Preparation of high production microorganisms B. Genetic manipulation of secondary metabolites producers IV. Obtaining new bioactive secondary metabolites A. Isolation from natural resources D B. Producers of bioactive compounds E C. Screening D. Semisynthetic and synthetic bioactive products E. Hybrid bioactive products and combinatorion biosynthesis V. Regulation of secondary metabolites production A.Growth phases of microbial culture B. Control of fermentation by basal nutrients C. How signals from the medium are received D. Regulation by low molecular compounds E. Autoregulators F. Regulation by metal ions VI. Resistance to secondary metabolites A. Resistance of bioactive secondary metabolites producers B. Resistance in pathogenic microorganisms VII. References

I. Introduction

C. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS

Medicine of twentieth century, especially its second half, was transformed by the discovery of antibiotics and other bioactive secondary metabolites produced by

microorganisms. Antibiotics are defined as microbial products that inhibit the growth of other microorganisms. After the antibacterial effect of penicillin had been observed by Fleming, a number of other antibiotics were discovered, mainly those produced by soil Streptomyces and moulds. Moreover, a broad spectrum of natural products having other effects on living organisms were found in microorganisms. In addition to standard antibiotics, the following compounds have also been found: coccidiostatics used in poultry farming, antiparasitic compounds with a broad spectrum of activity against nematodes and arthropods, substances with antitumor activity, immunosuppressors, thrombolytics (staphylokinase), compounds affecting blood pressure, end so forth. Microbial metabolites also exhibit good herbicide and pesticide activities and are biodegradable. However, microbial herbicides and pesticides only exceptionally used (e.g. bialaphos) due to their high price. Another special group of natural products are the enzyme inhibitors synthesized by microorganisms (Umezawa et al., 1976). These compounds can inhibit antibiotic derading enzymes, as well as certain enzyme activities in human metabolism that cause illness. Many enzyme inhibitors are protease inhibitors, variously active against pepsin, papain, trypsin, chymotrypsin, catepsin, elastase, renin, etc. Inhibitors of glucosidases, cyclic AMP phosphodiesterase, different carbohydrases, esterases, kinases, phosphatases, etc. have been also isolated from Streptomyces. The enzyme inhibitors that block synthesis of cholesterol are also important. Other exhibit the immunosuppressive effects, the most famous of them being cyclosporin A (a cyclic undecapeptide) produced by filamentous fungi. Some macrolide antibiotics, isolated from Streptomyces, are also immunosuppressives. Several thousands biologically active compounds have been deseribed and each year new compounds are isolated from microorganisms. Microorganisms are a virtually unlimited source of novel chemical structures with many potential therapeutic applications. The therm "secondary metabolite" used for some microbial products Bu´Lock (1961) and suitability of this therm discused Bennett and Bentley (1989). Secondary metabolites are meant compounds that the microorganism can synthesize but they are not essential for basic metabolic processes such as growth and reproduction. Nevertheless many secondary compounds function as the so-called signal molecules, used to control the producer’s metabolism. Another function attributed to antibiotics is a suppression of competing microorganisms in the environment whereby the antibiotic- producing microorganisms have an advantage in competing for nutrients with the other microorganisms. The production of secondary metabolites in microorganisms isolated from nature is rather low in most cases.To be usable for the commercial production of secondary metabolites, high yilding strains need to be selected through multiple mutations of the strain´s genetic material, optimization of culture conditions and genetic engineering.

II. Chemistry and biosynthesis

In spite of variety of their structures, bioactive secondary metabolites are synthesized from simple building units used in living organisms for the biosynthesis of cellular structures. These units include amino acids, acetate, propionate, sugars, nucleotides, etc.

According to their structure and type of biosynthesis, secondary metabolites are classified to form several groups.

A. PEPTIDE AND PEPTIDE-DERIVATIVE ANTIBIOTICS

Microorganisms produce a number of peptides as secondary metabolites. These peptide antibiotics are not synthetized on ribosomes but on enzyme complexes called peptide synthetases (Lipmann et al., 1971; Laland and Zimmer, 1973). In peptide antibiotic the peptide chain is often cyclic or branched. In addition to L-amino acids, other compounds can also be present in the molecule, such as D-amino acids, organic acids, pyrimidines and sugar molecules. The wellknown bioactive peptides, gramicidins and bacitracins are produced by different strains of Bacillus licheniformis and Bacillus brevis but some of them are produced by Streptomyces (Kleinkauf and von Doehren, 1986). The linear molecule of gramicidin A (Fig. 1) and the cyclic molecule of gramicidin S (Fig. 2) belong to the structurally simplest class of peptide antibiotics. Bacitracins are an example of cyclic peptides having a side chain (Fig. 3). In the molecule of bleomycin, the sugars L-glucose and 3-O-carbamoyl-D-mannose are found. Peptide antibiotics containing an atom of iron or phosphorus in the molecule have also been isolated. If two molecules of cysteine are present in the peptide antibiotic, they are linked by a sulfide bridge. The -CO-O- bond in the antibiotic molecule is present in lactones. Such antibiotics are represented especially by the group of actinomycins that contain a phenoxazine dicarboxylic group bearing two peptide chains. The enniatine molecule consists of three residues of branched amino acids, L-valine, L-leucine and L-isoleucine, and three residues of D-2-hydroxyisovaleric acid (D-Hyiv) (Billich and Zocher, 1987). The amino acids and D-Hyiv are linked by alternating amide and ester bonds. The amide bonds are finally N-methylated. Molecular conformation is important for the biological activity of peptide antibiotics. especially for the peptides capable of formating of chelates with metals. Studies showed three-dimensional molecular structures with many hydrogen bonds (Iitaka, 1978). In the + + case of valinomycin (L-Val-D-Hyiv-D-Val-L-Lac)3, which transports K and Rb ions across natural and artificial membranes, the molecule is symmetrical in three dimensions if it forms a complex with the metal. If it is not in the form of the complex, it has only a pseudocentral symmetry. The biosynthesis of peptide antibiotics takes place on a multienzyme complex. Kleinkauf and von Doehren,1983; Kleinkauf and von Doehren, 1986) The individual amino acids are activated using ATP to form aminoacyl adenylates. The aminoacyl groups are transferred to the enzyme thiol groups where they are bound as thioesters. The structural arrangement of the thiol groups in the synthetases determines the order of amino acids in the peptide. The formation of peptide bonds is mediated by 4- phosphopantetheine, an integral part of the multifunctional multienzyme. The intermediate peptides are also bound to the synthetases by the thioester bond. The way in which the order of the amino acids in the molecule is regulated is not known. It is probably determined by the tertiary configuration of the enzyme.

Our knowledge of the biosynthesis of peptide antibiotics comes mostly from the study of the gramicidin S and bacitracin synthetases. Gramicidin S synthetase consists of two complementary enzymes having molecular weights of 100 kD and 280 kD while bacitracin synthetase consists of three subunits (Roland et al., 1977) (Fig. 4) having molecular weights of 200, 210 and 360 kD (Ishiara et al., 1975). Each subunit contains phosphopantetheine. Enzyme A activates the first five amino acids of bacitracin, enzyme B activates L-Lys and L-Orn, and the enzyme C activates the other five amino acids. D-amino acids are produced by racemization of their L-forms directly on enzyme complex. Initiation and elongation start on subunit A up to the pentapeptide, independently of the presence of the subunits B and C. The pentapeptide is transferred to subunit B where two other amino acids are added. The heptapeptide is subsequently transferred to subunit C where the biosynthesis of bacitracin is finished. The cyclization is achieved by binding the asparagine carboxy group to the epsilon-amino group of lysine, whereas, the isoleucine carboxyl group is bound to the alpha-amino group of the same lysine (Laland et al., 1978). The antibiotic activity of bacitracin results in an efficient inhibition of proteosynthesis and cell wall synthesis but other effects such as an interference with cytoplasmic membrane components and cation-dependent antifungal effects have been observed as well. In the case of gramicidin S, hemolytic effects, inhibition of protein phosphatases and interaction with nucleotides have been observed in addition to the antibacterial activity. Even though antibiotics normally have several mechanisms of action, the primary one is defined to be the effect observed at the lowest active concentration. The peptide antibiotics are efficient mainly against Gram-positive bacteria. The b-lactams are peptide derived secondary metabolites. They are produced by different microorganisms . Several review sumarise reseach in these area (Martin and Liras, 1989; Jensen and Demain, 1995). The main producers are fungi (penicillins) but they are produced also by Strepromyces ( clavulanic acid) and Cephalosporium (cephalosporins). The main representatives of ß-lactams are penicillins and cephalosporins. Penicillins have a thiazoline ß-lactam ring in the molecule and differ, one from another, by side chains linked via the amino group (Fig. 5). Cephalosporins have a basic structure similar to that of penicillins and the derivatives are also formed by a variation of the side chain. The thiazolidine ß-lactam ring is synthesized using three amino acids: L-alpha-amino adipic acid, L-cystein and L-valine. By condensation of these three amino acids, a tripeptide is formed. It is transformed to the molecule of penicillin or cephalosporin through subsequent transformations (Fig. 6). Clavulanic acid, produced by Streptomyces clavuligerus, also belongs to ß- lactamfamily (Reading and Cole, 1977). This acid has a bicyclic ring structure resembling that of penicillin, except that oxygen replaces sulfur in the five-membered ring (Fig. 7.). Clavulanic acid is an irreversible inhibitor of many ß-lactamases. The discovery of clavulanic acid was a starting point for the development of penicillin analogues able to inactivate these enzymes. Penicillins are especially active against Gram-positive bacteria but some semisynthetic penicillins, such as ampicillin, that is lipophilic as compared to, for example, benzyl penicillin, are also effective against Gram-negative bacteria. This

effect is explained by their easier entering the cells of Gram-negative bacteria that have a high lipid content in the cell wall. ß-lactam antibiotics interfere with the synthesis of bacterial cell wall and thus inhibit bacterial growth. Such a mechanism of action does little harm to the macroorganism to which ß-lactams are applied. Another example of amino acid bioactive substances are the glycopeptides including semisynthetic derivatives (Zmijewski Jr. and Fayreman, 1995). The best known of all is vancomycin (Fig. 8) (Harris and Harris, 1982), effective against gram-positive bacteria. This antibiotic is widely used in medicine, especially against ß-lactam resistant strains. Vancomycin is not absorbed from the gastrointestinal tract and is used to treat enterocolitis caused mainly by Clostridium difficile. Vancomycin is produced by many species, of which Amycolotopsis orientalis is used for commercial production. Glycopeptides are composed of either seven modified or unusual aromatic amino acids or a mix of aromatic and aliphatic amino acids. By the substitution of amino acids in the amino acid core, derivatives of amino glycosides are formed. In vancomycin the aminosugar vancosamine is bound to the amino acid core. The removal of aminosugar reduces the activity of vancomycin two- to fivefold. The sugars seem to play an important role in imparting the enhanced pharmacokinetic properties for vancomycin-type, glycopeptide antibiotics.

B. POLYKETIDE-DERIVATIVES

Polyketides are a large group of secondary metabolites synthesized by decarboxylative condensation malonyl units often with subsequent cyclization of the polyketo chain . The starter group may be an acetate but also pyruvate, butyrate, ethyl malonate, paraaminobenzoic acid, etc. The formation of the initial polyketo chain is similar to that taking place during the biosynthesis of fatty acids, and is catalyzed by polyketide synthases. Simple carboxylic acids are activated as thioesters (acyl-SCoA) which are carboxylated to form malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA and after decarboxylation polymerized. ( Lynen, and Reichert, 1951; Lynen, 1959; Lynen and Tada, 1961). A principal role is played by the Acyl Carrier Protein (ACP) (Goldman and Vagelos, 1962). ACP detected throughout the growth of Streptomyces glaucescens was purified to homogenity and found to behave like many othes ACPs from bacteria and plants (Sumers et al. 1995). The ACP prosthetic group in many microorganisms is 4´-phosphopantothenic acid. Its terminal groups and acyls produced by polymerization are bound via the -SH group. The acyls are transferred to the other -SH group, that is a part of the cysteine molecule. Polyketide synthases have not yet been isolated and their properties have been deduced from the analyses of DNA sequences of cloned genes. Polyketide synthases include two distinct groups located either in domains on multifunctional proteins or present on individual, monofunctional proteins (McDaniel et al., 1993, Shen and Hutchinson, 1993). The structure and function of polyketide synthase in antibiotics overwie Robinson (1991) and Bentley and Bennett (1999). 6-Methyl salicylic acid (6MS) represents one of the simplest polyketides formed by condensation and subsequent aromatisation of one acetylCoA molecule and three malonylCoA molecules. This compound was isolated from Penicillium patulum (Bu ´Lock and Ryan, 1958). By other metabolic steps 6MS is transformed to produce a toxin

called patulin (Sekiguchi, 1983; Sekiguchi et al., 1983). The synthesis of 6MS takes place on an enzymatic complex called 6MS synthetase (Fig. 9) (Dimroth et al., 1970,1976). The chemical structure of sometypical tetracyclines is shown in Fig. 10 and their biosynthesis in Figs. 11 and 12 (McCormick, 1965). Chlortetracycline (CTC) and tetracycline (TC) are produced by the actinomycete Streptomyces aureofaciens, whereas oxytetracycline (OTC) and tetracycline by the actinomycete Streptomyces rimosus. For a more extensive coverage of research, articles by Běhal et al. (1983), Běhal (1987) and Běhal and Hunter (1995) should be consulted.

Tetracyclines act as inhibitors of proteosynthesis. They are considered to be wide- spectrum antibiotics that are efficient against both Gram-positive and Gram-negative bacteria. However, having significant side effects on the human macroorganism, they are preferably used only in the case where other, less toxic antibiotics are not effective. Anthracyclines are synthesized in a similar way as tetracyclines, however, they often have one or several sugar residues in the molecule. Most often deoxy-sugars, synthesized from glucose, are present in the anthracycline molecule. Daunorubicin and doxorubicin (adriamycin) (Fig. 13) are excellent antitumor agents, which are widely used in the treatment of a number of solid tumors and leukemias in human. Unfortunately, these drugs have dose limiting toxicities such as cardiac damage and bone marrow inhibition. In recent years, a variety of drug delivery systems for anthracyclines have been reported. In most cases, the drugs were linked to high molecular compounds such as dextran (Levi-Schaff et al., 1982; Tanaka, 1994), DNA (Campeneere, 1979), and others. Anthracyclines are produced by many Streptomyces (Grein, 1987) and genetics of their production is well elaborated (Hutchinson, 1995). Macrolides are usually classified to include: proper macrolides having 12-, 14- or 16-membered macrocyclic lactone ring to which at least one sugar is bound, and polyenes having 26- to 38-atom lactone ring containing 2 to 7 unsaturated bonds. Besides the sugars bound to the lactone ring, an additional aromatic part is normally present in the polyene molecule. Both macrolides and polyenes are biosynthesized in the same way using identical building units. Macrolides represent a broad group of compounds and new substances have been incessantly added to the list. Macrolides usually possess an antibacterial activity whereas polyens are mostly fungicides. Erythromycins produced by Saccharopolyspora erythrea (Fig. 14), together with oleandomycin and picromycin, belong to the best known 14-membered lactone ring macrolides (Harris et al., 1965). Macrolides with a 16-membered ring are represented by tylosin (Fig. 15) (Omura et al., 1975), that is produced by Streptomyces fradiae , as well as by leucomycin, spiramycin, etc. The synthesis of lactone ring is similar to that observed in the case of other polyketides. In contrast to aromatics, pyruvate and butyrate units are more often used in the biosynthesis, instead of acetate ones. The greatest difference, however, consists in the fact that, instead of aromatic rings, a lactone ring is formed. Keto- and methyl groups of the polyketide chain, from which macrolides are formed, are normally transformed more frequently. Nystatin is the best known polyene secondary metabolite (Fig. 16). Candicidine is another well known secondary metabolite belonging to the polyene group. Its molecule

includes p-aminoacetophenone as the terminal group. 4-amino benzoic acid (PABA) was identified as a precursor of the aromatic part of candicidine molecule (Liu et al., 1972, Martin, 1977). The sugars found in macrolide and polyene molecules are not usuallyencountered in microbial cells. They include both basic and neutral sugar molecules and L-forms are often found. So far, at least 15 different sugars have been described to occur in macrolides and polyenes. All of them are 6-deoxy sugars; some of them are N- methylated, others have the methyl on either the oxygen or carbon atom. As it has been repeatedly proven (Corcoran and Chick, 1966), glucose is primarily incorporated into macrolide sugar residues. Also in Streptomyces griseus, glucose, mannose and galactose were incorporated to a greater extent into the mycosamine candicidine, as compared to its aglycon (Martin and Gil, 1979). The transformation of glucose to a corresponding sugar takes place in the form of the nucleoside diphosphate derivatives, which is similar to the situation found in the case of other secondary metabolites. Avermectins consist of a 16-membered, macrocyclic lactone to which the disaccharide oleandrose is bound (Fig. 17) (Burg, R.W., 1979; Miller, T.W., 1979). Avermectins are produced by Streptomyces avermitillis. The macrocyclic ring of avermectins is synthesized, as other polyketides, by producing a chain from acetate, propionate and butyrate building units. Oleandrose (2,6-dideoxy-3-O-methylated hexose) is synthesized from glucose. Avermectins are potent antiparasitic compounds active against a broad spectrum nematode and anthropod parasites. They lack antifungal and antibacterial activities. They bind to a specific, high-affinity site present in nematodes but not in vertebrates. Its dosage for animal and human is extremely low. Ivermectin (22,23-dihydroavermectin B1) is a semisynthetic compound which is used to control internal and external parasites in animals and is the most potent anthelmintic compound of all. Avermectins are also employed in human medicine and plant protection. Detailed reviews on the uses and biosynthesis of avermectins can be found in recent monographs (MacNeil, 1995; Ikeda and Omura, 1995). Polyethers form a large group of structural related natural products mainly produced by Streptomyces (Birch and Robinson, 1995). They are potent coccidiostats (monensin, salinomycin) and are used in the agricultural arena.(Westley, 1977). Polyethers are compouns possesing the ability to form lipid-soluble complexes that provide a vehicle for a wide variety of cations to traverse lipid barrieres. This ion- bearing property led to their being named ionophores (Moore and Pressman, 1994). Backbones of polyethers are synthetized from acetate, propionate and butyrate (monensin A) units. Isobutyrate and n-butyrate are efficiently incorporated into polyether antibiotics (Pospíšil et al., 1983). Incorporation of isobutyrate was explained by formal conversion of isobutyryl-CoA into n-butyryl-CoA or methylmalonyl-CoA by isobutyryl-CoA mutase and methylmalonyl-CoA mutase, respectively.

C. OTHER GROUPS OF BIOACTIVE PRODUCTS

Chloramphenicol (Fig. 18) is produced by Streptomyces venezuelae (Vining and Westlake, 1984). At present, however, the antibiotic is commercially produced using a

fully synthetic process. In contrast to polyketides, the aromatic ring of chloramphenicol molecule is synthesized from glucose via chorismic acid and p-amino benzoic acid in the microbe. Streptomycin (Fig. 19) is a well-known aminoglycoside antibioticoriginaly discovered by Selnon Waksman. It is synthesized by many streptomycetes to produce a number of derivatives. The molecule of streptomycin consists of three components: streptidine, L-streptose and N-methyl-L-glucosamine. None of these components has been found in the primary metabolism of microorganisms. The steps of streptomycin biosynthesis were disclosed mainly by Walker (Walker and Walker, 1971), who also studied the relevant enzymes (Walker, 1975). The importance of streptomycin consists mainly in its ability to suppress Mycobacterium tuberculosis, resulting in effective suppression of tuberculosis, especially in developed countries. Bialaphos is formed from two L-alanine residues and the amino acid phosphinothricine. The latter compound is synthesized by streptomycetes from acetylCoA and phosphoenolpyruvate, and subsequently methylated using methionine as the methyl donor (Bayer et al., 1972; Ogawa et al., 1973). The producing microorganisms are Streptomyces hygroscopicus and Streptomyces viridochromogenes. Bialaphos, as well as phosphinothricine, inhibits the activity of glutamine synthetase.

III. Genetics and molecular genetics

A. PREPARATION OF HIGH PRODUCTION MICROORGANISMS

The structural genes encoding the enzymes that synthesize secondary metabolites are mostly located on chromosomes They are often organized in gene clusters (Binnie et al., 1989; Malpartida and Hopwood, 1984; Lotvin et al., 1992; Martin, 1992). Resistance of the producer to its own products are located either at the beginning or at the end of the cluster, often in both positions. In addition to the resistance and structural genes, regulatory genes are important in secondary metabolites production, however, they function is poorly understand. Microorganisms that are isolated from nature (wild type strains) produce small amounts of secondary metabolites. Sometimes during selection and subsequent cultivation in the laboratory, a changes occur, making the cultivated strain non-identical with the original strain. In such cases it should be remarked that the term wild type strain only refers to the fact that the strain did not undergo an “artificial” genetic change. In order for the commercial production of secondary metabolites could be profitable, higher levels of the secondary metabolites synthesis are reached via genetic changes of producers. Mutants are isolated by exposure of spores to UV irradiation, X-rays, γ-rays, α-particles or chemical mutagens (nitrogen mustards, N-methyl-N´-nitro-N-nitroso guanidine). Combined mutagenesis using various mutagens is often used. The surviving spores give rise to individual colonies of isolates, whose capability of secondary metabolites production is then tested. Mutants that exhibit poor growth and sporulation ability are not suitable candidates for further improvement, even if their secondary

metabolites production may exceed that of the original strain. Today’s high production strains, that synthesize as high as 10 000-fold levels of secondary metabolites, compared to the original strains, are the result of many year of costly strain improvement. Unfortunately, these high production strains can revert to lose their overproduction though spontaneous mutagenesis. When high production strains are prepared by mutagenesis, a type of mutant that loses some of the structural genes can also be obtained. Such a mutant can exhibit a higher level of a secondary metabolite intermediate whose transformation stopped due to the absence of the corresponding enzyme. By crossing these mutants, some biosynthetic pathways used to synthesize secondary metabolites were elucidated, e.g. tetracyclines (McCormick et all.1960). Loss of the capability of secondary metabolite production in the strains where extrachromosomal DNA was removed (e.g. by using acriflavine or ethidium bromide) suggests that the regulatory genes are located on plasmids (Hotta et al, 1977; Okanishi, 1979; Akagava et al., 1979; Boronin et al., 1974; Ikeda et al., 1982).

B. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

Structural genes for a number of secondary metabolites have been cloned into host microorganisms. Similarly, genes for secondary metabolites resistance and other regulatory genes have also been cloned. Streptomyces lividans was found to be a suitable acceptor of foreign genetic material, in which a low degree of restriction of this genetic material exists. This microorganism can host various plasmids and phage vectors. However, at the same time, this microorganism was found not to be usable for the synthesis of various secondary metabolites or of their high levels. The secondary metabolites biosynthesis is a very complex process that requires not only the structural genes for ESM but also the genes for regulation of their biosynthesis. Moreover, the overproduction of a secondary metabolite has to be coordinated with the primary metabolism of the producing microorganism. The cloning of structural genes and genes for resistance to the own secondary metabolite enables us to work out genetic maps of the producers. On the basis of those maps, hybrid clusters combined of two and more clusters of different secondary metabolites can be created. Consequently, semisynthetic secondary metabolites can be produced that may possess new biological activities or an antibiotic activity against resistant strains. Polyketide synthase genes of microorganisms producing various polyketides have also been hybridized (Hopwood and Sherman, 1990). As a result, a great similarity of polyketide synthases from various streptomycetes was evidenced (Malpartida et al., 1987; Butler et al., 1990).

IV. Obtaining new bioactive secondary metabolites

A. ISOLATION FROM NATURAL RESOURCES

In spite of the fact that several thousands of compounds isolated from microorganisms having some biological activity are known, new substances are still saught by pharmaceutical companies. The probability of finding a new compound that would be usable as a new antibiotic or another biologically active compound is low, so a great number of microorganisms have to be screened. A rough estimation says that about 100 000 microorganisms are screened for the presence of biologically active compounds per year. Modern screens are highly automated. The selection methods used, the targets, and the methods of detection of the biological activity are normally not published. Preparation of a new biologically active compound and its introduction into clinical practice requires the cooperation of scientists from various scientific disciplines and years of clinical trials. This effort can be divided into three parts:(Yarbrough et al., 1993): 1. microbiology -collection of source samples (soil) -isolation of diverse microbes -fermentation to enhance diversity -reproduce fermentation -enhance the production for isolation -taxonomy of the organism 2. molecular biology/pharmacology -target selection -screen design/implementation -high through-put screening -identification of active compounds -efficacy studies -mechanism of action 3. chemistry -active compound identification -characterisation/dereplication -isolation/purification -structure elucidation.

B. PRODUCERS OF BIOACTIVE COMPOUNDS

About 70 % of the known bioactive substances are produced by Streptomyces and the rest mainly by moulds and non-filamentous bacteria. With an increasing spectrum of efficiency of microbial metabolites, new, non-traditional sources of such compounds have been tappede. These include the microorganisms living under extreme conditions (high and low temperatures, etc.), sea living microorganisms, and multicelular plants and animals. Another important source of new compounds are the mutants of producers of known active substances, e.g. blocked mutants.

D. SCREENING

The enterprise of screening microbial metabolites for new leads, first exploited by antibiotic researchers and today expanded to virtually all fields of therapeutic interest, has proven successful and will continue as an important avenue to new drug discovery. The original method for determination of antibiotic efficiency consisted of the application of test extract to wells made in agar medium layer in Petri dishes to which the sensitive (target) microorganism was inoculated. Most often Staphylococcus aureus, Sarcina lutea, Klebsiella pneumoniae, Salmonella gallinarium, Pseudomonas spp., Bacillus subtilis, and Candida albicans were used. In case a compound with an antibiotic activity towards the testing microorganism was put into the well, it diffused through the agar medium and a halo was formed around the well, as a result of the suppressed growth of the microorganism. This classic plate assay has been modified and improved in many ways. The tests of other biological activities require different and frequently sophisticated methods. This is true especially when enzyme inhibitors are a case in point. Thus, Ogawara et al. (1986) chose a tyrosine protein kinase associated with the malignant transformation of the cell caused by retroviruses as the target in a biochemical screen, they found genistein, an isoflavone from Pseudomonas, exhibiting a specific inhibitory activity. Production of target enzymes using recombinant DNA methodology has dramatically expanded the number of potential targets that can be feasibly screened. A screen for the inhibitors of HIV reverse transcriptase is an example. The enzyme was produced in Escherichia coli, purified by affinity chromatography, and used to test natural products for the activity (Take et al., 1989).

D. SEMISYNTHETIC AND SYNTHETIC BIOACTIVE PRODUCTS

Natural products can be modified in various ways. The unspecificity of the enzyme systems facilitates the synthesis of certain secondary metabolites though the addition of selectedprecursors to the growth medium. Thus, the reaction equilibrium can be shifted to promote the production of the derivative required, e.g. the prepareation of penicillins with different side chains. The individual derivatives of penicillin and cephalosporin have slightly different antimicrobial spectra and are active against microorganisms resistant to other derivatives. The structuure of polypeptide antibiotics can also be modified by the addition of amino acids to the growth Replacement of a part of the metabolite molecule can be accomplished chemically or enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines and other antibiotics can be prepared. The production of semisynthetic penicillins and cephalosporins is facilitated by the fact that 6-amino penicillanic and 7-amino cephalosporanic acids are easily prepared. The side chain is removed by the action of an enzyme or by a chemical hydrolysis (Fig. 20) then another acyl is bound chemically or enzymatically to the amino group in position 6 (penicillins) or 7 (cephalosporins). Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline, exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared to the original tetracyclines.

New derivatives of aminoglycosides also have been obtained by chemical and enzymatic modifications. As the majority of bioactive products have rather complex structures, their chemical synthesis is mostly more expensive than the production by fermentation. An exception to the rule seems to be chloramphenicol, that is normally prepared using a chemical synthesis.

E. HYBRID BIOACTIVE PRODUCTS

Genetic engineering methods have recently advanced so much that now we can suitably combine structural genes of two or even more bioactive secondary metabolites producers. If these genes are expressed, a hybrid bioactive products is synthesized, ore that cannot be found in nature (Hutchinson, 1987, 1988; Tomich, 1988; Hopwood, 1993). Hopwood et al. (1985, 1986,) used this method with the genes of actinorhodin synthesis and obtained related hybrid macrolides, mederhodin A and B, dihydromederhodin A and dihydrogranatirhodin. A new anthracyclines were produced when a DNA segment was cloned from Streptomyces purpurascenc ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase Streptomyces galilaeus ATCC 31615 (Niemi et al., 1994).

V. Regulation of secondary metabolites production

A. GROWTH PHASES OF Stepromyces

In the cultures of Streptomyces capable of secondary metabolite production several growth phases representing different physiological statescan be distinguished:

5. Preparatory phase (lag phase) - the biomass increase is low, the culture is adapting to the new environment. 6. Growth phase (the term logarithmic phase is not suitable for most Streptomyces since their growth curves are not exponential functions) - intensive growth is taking place, accompanied by a low secondary metabolite synthesis. This phase is roughly equivalent to "trophophase". 7. Transition phase - characterized by a decreased growth rate; the secondary metabolite production is started. The enzymes of secondary metabolism are synthesized (Běhal, 1986a; Běhal, 1986b) and proteosynthesis slowed down. 8. Production phase - characterized by a significant reduction of the growth rate (sometimes growth is even completely ceased), a negligible change in the biomass concentration, and an intensive synthesis of the secondary metabolite. This phase is some times called "idiophase".