Bioactive Products From Streptomyces

7/31/2019 Bioactive Products From Streptomyces

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Adv. Appl. Microbiol. 47, 113-156, 2000.

Bioactive Products fromStreptomyces

VLADISLAV B HALInstitute of Microbiology

Academy of Sciences of the Czech RepublicPrague, Czech Republic

I. IntroductionII. Chemistry and biosynthesis

A. Peptide and peptide-derivative antibioticsB. Polyketide derivativesC. Other groups of bioactive products

III. Genetics and molecular geneticsA. Preparation of high production microorganismsB. Genetic manipulation of secondary metabolites producers

IV. Obtaining new bioactive secondary metabolitesA. Isolation from natural resourcesB B. Producers of bioactive compoundsC C. ScreeningD. Semisynthetic and synthetic bioactive productsE. Hybrid bioactive products and combinatorion biosynthesis

V. Regulation of secondary metabolites productionA.Growth phases of microbial cultureB. Control of fermentation by basal nutrientsC. How signals from the medium are receivedD. Regulation by low molecular compoundsE. AutoregulatorsF. Regulation by metal ions

VI. Resistance to secondary metabolitesA. Resistance of bioactive secondary metabolites producersB. Resistance in pathogenic microorganisms

VII. References

I. Introduction

A. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS


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Medicine of twentieth century, especially its second half, wastransformed by the discovery of antibiotics and other bioactivesecondary metabolites produced by microorganisms. Antibiotics are

defined as microbial products that inhibit the growth of othermicroorganisms. After the antibacterial effect of penicillin had beenobserved by Fleming, a number of other antibiotics were discovered,mainly those produced by soil Streptomyces and moulds. Moreover, abroad spectrum of natural products having other effects on livingorganisms were found in microorganisms. In addition to standardantibiotics, the following compounds have also been found:coccidiostatics used in poultry farming, antiparasitic compounds witha 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 andpesticide activities and are biodegradable. However, microbialherbicides and pesticides only exceptionally used (e.g. bialaphos) dueto their high price.

Another special group of natural products are the enzymeinhibitors synthesized by microorganisms (Umezawa et al., 1976).These compounds can inhibit antibiotic derading enzymes, as well ascertain enzyme activities in human metabolism that cause illness.Many enzyme inhibitors are protease inhibitors, variously activeagainst pepsin, papain, trypsin, chymotrypsin, catepsin, elastase,

renin, etc. Inhibitors of glucosidases, cyclic AMP phosphodiesterase,different carbohydrases, esterases, kinases, phosphatases, etc. havebeen also isolated from Streptomyces. The enzyme inhibitors thatblock synthesis of cholesterol are also important. Other exhibit theimmunosuppressive effects, the most famous of them beingcyclosporin A (a cyclic undecapeptide) produced by filamentousfungi. Some macrolide antibiotics, isolated from Streptomyces, arealso immunosuppressives.

Several thousands biologically active compounds have beendeseribed and each year new compounds are isolated frommicroorganisms. Microorganisms are a virtually unlimited source of

novel chemical structures with many potential therapeuticapplications.The therm "secondary metabolite" used for some microbial

products BuLock (1961) and suitability of this therm discusedBennett and Bentley (1989). Secondary metabolites are meantcompounds that the microorganism can synthesize but they are notessential for basic metabolic processes such as growth andreproduction. Nevertheless many secondary compounds function asthe so-called signal molecules, used to control the producersmetabolism. Another function attributed to antibiotics is asuppression of competing microorganisms in the environment

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whereby the antibiotic-producing microorganisms have an advantagein 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 thecommercial production of secondary metabolites, high yilding strainsneed to be selected through multiple mutations of the strainsgenetic material, optimization of culture conditions and geneticengineering.

II. Chemistry and biosynthesis

In spite of variety of their structures, bioactive secondarymetabolites are synthesized from simple building units used in livingorganisms for the biosynthesis of cellular structures. These unitsinclude amino acids, acetate, propionate, sugars, nucleotides, etc.According to their structure and type of biosynthesis, secondarymetabolites are classified to form several groups.

A. PEPTIDE AND PEPTIDE-DERIVATIVE ANTIBIOTICS

Microorganisms produce a number of peptides as secondarymetabolites. These peptide antibiotics are not synthetized onribosomes 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 toL-amino acids, other compounds can also be present in the molecule,such as D-amino acids, organic acids, pyrimidines and sugarmolecules. The wellknown bioactive peptides, gramicidins andbacitracins are produced by different strains ofBacillus licheniformisand 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 moleculeof gramicidin S (Fig. 2) belong to the structurally simplest class ofpeptide antibiotics. Bacitracins are an example of cyclic peptideshaving a side chain (Fig. 3). In the molecule of bleomycin, the sugars

L-glucose and 3-O-carbamoyl-D-mannose are found. Peptideantibiotics containing an atom of iron or phosphorus in the moleculehave also been isolated. If two molecules of cysteine are present inthe peptide antibiotic, they are linked by a sulfide bridge. The -CO-O-bond in the antibiotic molecule is present in lactones. Such antibioticsare represented especially by the group of actinomycins that containa phenoxazine dicarboxylic group bearing two peptide chains.

The enniatine molecule consists of three residues of branchedamino acids, L-valine, L-leucine and L-isoleucine, and three residuesof D-2-hydroxyisovaleric acid (D-Hyiv) (Billich and Zocher, 1987). The

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amino acids and D-Hyiv are linked by alternating amide and esterbonds. 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 ofchelates with metals. Studies showed three-dimensional molecularstructures 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 issymmetrical 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 pseudocentralsymmetry.

The biosynthesis of peptide antibiotics takes place on amultienzyme complex. Kleinkauf and von Doehren,1983; Kleinkauf

and von Doehren, 1986) The individual amino acids are activatedusing ATP to form aminoacyl adenylates. The aminoacyl groups aretransferred to the enzyme thiol groups where they are bound asthioesters. The structural arrangement of the thiol groups in thesynthetases determines the order of amino acids in the peptide. Theformation of peptide bonds is mediated by 4-phosphopantetheine, anintegral part of the multifunctional multienzyme. The intermediatepeptides are also bound to the synthetases by the thioester bond.

The way in which the order of the amino acids in the molecule isregulated is not known. It is probably determined by the tertiaryconfiguration of the enzyme.

Our knowledge of the biosynthesis of peptide antibiotics comesmostly from the study of the gramicidin S and bacitracinsynthetases.Gramicidin S synthetase consists of two complementary enzymeshaving molecular weights of 100 kD and 280 kD while bacitracinsynthetase 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 Aactivates the first five amino acids of bacitracin, enzyme B activatesL-Lys and L-Orn, and the enzyme C activates the other five amino

acids. D-amino acids are produced by racemization of their L-formsdirectly on enzyme complex. Initiation and elongation start onsubunit A up to the pentapeptide, independently of the presence ofthe subunits B and C. The pentapeptide is transferred to subunit Bwhere two other amino acids are added. The heptapeptide issubsequently transferred to subunit C where the biosynthesis ofbacitracin is finished. The cyclization is achieved by binding theasparagine carboxy group to the epsilon-amino group of lysine,whereas, the isoleucine carboxyl group is bound to the alpha-aminogroup of the same lysine (Laland et al., 1978).

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The antibiotic activity of bacitracin results in an efficient inhibitionof proteosynthesis and cell wall synthesis but other effects such asan interference with cytoplasmic membrane components and cation-

dependent antifungal effects have been observed as well. In the caseof gramicidin S, hemolytic effects, inhibition of protein phosphatasesand interaction with nucleotides have been observed in addition tothe antibacterial activity. Even though antibiotics normally haveseveral mechanisms of action, the primary one is defined to be theeffect observed at the lowest active concentration. The peptideantibiotics are efficient mainly against Gram-positive bacteria.The b-lactams are peptide derived secondary metabolites. They areproduced by different microorganisms . Several review sumarisereseach in these area (Martin and Liras, 1989; Jensen and Demain,1995). The main producers are fungi (penicillins) but they areproduced also by Strepromyces (clavulanic acid) andCephalosporium (cephalosporins). The main representatives of -lactams are penicillins and cephalosporins. Penicillins have athiazoline -lactam ring in the molecule and differ, one from another,by side chains linked via the amino group (Fig. 5). Cephalosporinshave a basic structure similar to that of penicillins and the derivativesare also formed by a variation of the side chain.

The thiazolidine -lactam ring is synthesized using three aminoacids: L-alpha-amino adipic acid, L-cystein and L-valine. Bycondensation of these three amino acids, a tripeptide is formed. It is

transformed to the molecule of penicillin or cephalosporin throughsubsequent transformations (Fig. 6).Clavulanic acid, produced by Streptomyces clavuligerus, also

belongs to -lactamfamily (Reading and Cole, 1977). This acid has abicyclic ring structure resembling that of penicillin, except thatoxygen replaces sulfur in the five-membered ring (Fig. 7.). Clavulanicacid is an irreversible inhibitor of many -lactamases. The discoveryof clavulanic acid was a starting point for the development ofpenicillin analogues able to inactivate these enzymes.

Penicillins are especially active against Gram-positive bacteria butsome semisynthetic penicillins, such as ampicillin, that is lipophilic as

compared to, for example, benzyl penicillin, are also effective againstGram-negative bacteria. This effect is explained by their easierentering the cells of Gram-negative bacteria that have a high lipidcontent in the cell wall. -lactam antibiotics interfere with thesynthesis of bacterial cell wall and thus inhibit bacterial growth. Sucha mechanism of action does little harm to the macroorganism towhich -lactams are applied.Another example of amino acid bioactive substances are theglycopeptides including semisynthetic derivatives (Zmijewski Jr. andFayreman, 1995). The best known of all is vancomycin (Fig. 8) (Harris

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and Harris, 1982), effective against gram-positive bacteria. Thisantibiotic is widely used in medicine, especially against -lactamresistant strains. Vancomycin is not absorbed from the

gastrointestinal tract and is used to treat enterocolitis caused mainlyby Clostridium difficile.Vancomycin is produced by many species, of whichAmycolotopsis

orientalis is used for commercial production. Glycopeptides arecomposed of either seven modified or unusual aromatic amino acidsor a mix of aromatic and aliphatic amino acids. By the substitution ofamino acids in the amino acid core, derivatives of amino glycosidesare formed. In vancomycin the aminosugar vancosamine is bound tothe amino acid core. The removal of aminosugar reduces the activityof vancomycin two- to fivefold. The sugars seem to play an importantrole in imparting the enhanced pharmacokinetic properties forvancomycin-type, glycopeptide antibiotics.

B. POLYKETIDE-DERIVATIVES

Polyketides are a large group of secondary metabolites synthesizedby decarboxylative condensation malonyl units often with subsequentcyclization of the polyketo chain . The starter group may be anacetate but also pyruvate, butyrate, ethyl malonate,paraaminobenzoic acid, etc. The formation of the initial polyketochain is similar to that taking place during the biosynthesis of fatty

acids, and is catalyzed by polyketide synthases. Simple carboxylicacids are activated as thioesters (acyl-SCoA) which are carboxylatedto form malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA and afterdecarboxylation polymerized. ( Lynen, and Reichert, 1951; Lynen,1959; Lynen and Tada, 1961). A principal role is played by the AcylCarrier Protein (ACP) (Goldman and Vagelos, 1962). ACP detectedthroughout the growth ofStreptomyces glaucescens was purified tohomogenity and found to behave like many othes ACPs from bacteriaand plants (Sumers et al. 1995). The ACP prosthetic group in manymicroorganisms is 4-phosphopantothenic acid. Its terminal groupsand acyls produced by polymerization are bound via the -SH group.

The acyls are transferred to the other -SH group, that is a part of thecysteine molecule. Polyketide synthases have not yet been isolatedand their properties have been deduced from the analyses of DNAsequences of cloned genes. Polyketide synthases include two distinctgroups located either in domains on multifunctional proteins orpresent on individual, monofunctional proteins (McDaniel et al.,1993, Shen and Hutchinson, 1993). The structure and function ofpolyketide synthase in antibiotics overwie Robinson (1991) andBentley and Bennett (1999).

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6-Methyl salicylic acid (6MS) represents one of the simplestpolyketides formed by condensation and subsequent aromatisation ofone acetylCoA molecule and three malonylCoA molecules. This

compound was isolated from Penicillium patulum (BuLock and Ryan,1958). By other metabolic steps 6MS is transformed to produce atoxin called patulin (Sekiguchi, 1983; Sekiguchi et al., 1983). Thesynthesis of 6MS takes place on an enzymatic complex called 6MSsynthetase (Fig. 9) (Dimroth et al., 1970,1976).

The chemical structure of sometypical tetracyclines is shown inFig. 10 and their biosynthesis in Figs. 11 and 12 (McCormick, 1965).Chlortetracycline (CTC) and tetracycline (TC) are produced by theactinomycete Streptomyces aureofaciens, whereas oxytetracycline(OTC) and tetracycline by the actinomycete Streptomyces rimosus.For a more extensive coverage of research, articles by B hal et al. (1983), B hal (1987) and B hal and Hunter (1995) should be consulted.

Tetracyclines act as inhibitors of proteosynthesis. They areconsidered to be wide-spectrum antibiotics that are efficient againstboth Gram-positive and Gram-negative bacteria. However, havingsignificant side effects on the human macroorganism, they arepreferably used only in the case where other, less toxic antibioticsare not effective.

Anthracyclines are synthesized in a similar way as tetracyclines,

however, they often have one or several sugar residues in themolecule. Most often deoxy-sugars, synthesized from glucose, arepresent in the anthracycline molecule. Daunorubicin and doxorubicin(adriamycin) (Fig. 13) are excellent antitumor agents, which arewidely used in the treatment of a number of solid tumors andleukemias in human. Unfortunately, these drugs have dose limitingtoxicities such as cardiac damage and bone marrow inhibition. Inrecent years, a variety of drug delivery systems for anthracyclineshave been reported. In most cases, the drugs were linked to highmolecular 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 oftheir production is well elaborated (Hutchinson, 1995).

Macrolides are usually classified to include: proper macrolideshaving 12-, 14- or 16-membered macrocyclic lactone ring to which atleast one sugar is bound, and polyenes having 26- to 38-atom lactonering containing 2 to 7 unsaturated bonds. Besides the sugars boundto the lactone ring, an additional aromatic part is normally present inthe polyene molecule. Both macrolides and polyenes arebiosynthesized in the same way using identical building units.Macrolides represent a broad group of compounds and new

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substances have been incessantly added to the list. Macrolidesusually possess an antibacterial activity whereas polyens are mostlyfungicides.

Erythromycins produced by Saccharopolyspora erythrea (Fig.14), together with oleandomycin and picromycin, belong to the bestknown 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 caseof other polyketides. In contrast to aromatics, pyruvate and butyrateunits are more often used in the biosynthesis, instead of acetateones. The greatest difference, however, consists in the fact that,instead of aromatic rings, a lactone ring is formed. Keto- and methylgroups 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 belongingto the polyene group. Its molecule includes p-aminoacetophenone asthe terminal group. 4-amino benzoic acid (PABA) was identified as aprecursor of the aromatic part of candicidine molecule (Liu et al.,1972, Martin, 1977). The sugars found in macrolide and polyene molecules are notusuallyencountered in microbial cells. They include both basic and

neutral sugar molecules and L-forms are often found. So far, at least15 different sugars have been described to occur in macrolides andpolyenes. All of them are 6-deoxy sugars; some of them are N-methylated, others have the methyl on either the oxygen or carbonatom. As it has been repeatedly proven (Corcoran and Chick, 1966),glucose is primarily incorporated into macrolide sugar residues. Alsoin Streptomyces griseus, glucose, mannose and galactose wereincorporated to a greater extent into the mycosamine candicidine, ascompared to its aglycon (Martin and Gil, 1979). The transformation ofglucose to a corresponding sugar takes place in the form of thenucleoside 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 Streptomycesavermitillis. The macrocyclic ring of avermectins is synthesized, asother polyketides, by producing a chain from acetate, propionate andbutyrate building units. Oleandrose (2,6-dideoxy-3-O-methylatedhexose) is synthesized from glucose.

Avermectins are potent antiparasitic compounds active against abroad spectrum nematode and anthropod parasites. They lack

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antifungal and antibacterial activities. They bind to a specific, high-affinity site present in nematodes but not in vertebrates. Its dosagefor animal and human is extremely low. Ivermectin (22,23-

dihydroavermectin B1) is a semisynthetic compound which is used tocontrol internal and external parasites in animals and is the mostpotent anthelmintic compound of all. Avermectins are also employedin human medicine and plant protection. Detailed reviews on theuses and biosynthesis of avermectins can be found in recentmonographs (MacNeil, 1995; Ikeda and Omura, 1995).

Polyethers form a large group of structural related naturalproducts mainly produced by Streptomyces (Birch and Robinson,1995). They are potent coccidiostats (monensin, salinomycin) and areused in the agricultural arena.(Westley, 1977). Polyethers arecompouns possesing the ability to form lipid-soluble complexes thatprovide a vehicle for a wide variety of cations to traverse lipidbarrieres. This ion-bearing property led to their being namedionophores (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(Pospil et al., 1983). Incorporation of isobutyrate was explained byformal conversion of isobutyryl-CoA into n-butyryl-CoA ormethylmalonyl-CoA by isobutyryl-CoA mutase and methylmalonyl-CoA mutase, respectively.

C. OTHER GROUPS OF BIOACTIVE PRODUCTS

Chloramphenicol (Fig. 18) is produced by Streptomycesvenezuelae (Vining and Westlake, 1984). At present, however, theantibiotic is commercially produced using a fully synthetic process. Incontrast to polyketides, the aromatic ring of chloramphenicolmolecule is synthesized from glucose via chorismic acid and p-aminobenzoic acid in the microbe.

Streptomycin (Fig. 19) is a well-known aminoglycosideantibioticoriginaly discovered by Selnon Waksman. It is synthesizedby many streptomycetes to produce a number of derivatives. Themolecule of streptomycin consists of three components: streptidine,L-streptose and N-methyl-L-glucosamine. None of these componentshas been found in the primary metabolism of microorganisms. Thesteps 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 tosuppress Mycobacterium tuberculosis, resulting in effectivesuppression of tuberculosis, especially in developed countries.

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Bialaphos is formed from two L-alanine residues and the aminoacid phosphinothricine. The latter compound is synthesized bystreptomycetes from acetylCoA and phosphoenolpyruvate, and

subsequently methylated using methionine as the methyl donor(Bayer et al., 1972; Ogawa et al., 1973). The producingmicroorganisms are Streptomyces hygroscopicus and Streptomycesviridochromogenes. Bialaphos, as well as phosphinothricine, inhibitsthe activity of glutamine synthetase.

III. Genetics and molecular genetics

A. PREPARATION OF HIGH PRODUCTION MICROORGANISMS

The structural genes encoding the enzymes that synthesizesecondary metabolites are mostly located on chromosomes They areoften organized in gene clusters (Binnie et al., 1989; Malpartida andHopwood, 1984; Lotvin et al., 1992; Martin, 1992). Resistance of theproducer to its own products are located either at the beginning or atthe end of the cluster, often in both positions. In addition to theresistance and structural genes, regulatory genes are important insecondary metabolites production, however, they function is poorlyunderstand.

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 changesoccur, making the cultivated strain non-identical with the originalstrain. In such cases it should be remarked that the term wild typestrain only refers to the fact that the strain did not undergo anartificial genetic change.

In order for the commercial production of secondary metabolitescould be profitable, higher levels of the secondary metabolitessynthesis are reached via genetic changes of producers. Mutants areisolated by exposure of spores to UV irradiation, X-rays, -rays, - particles or chemical mutagens (nitrogen mustards, N-methyl-N-

nitro-N-nitroso guanidine). Combined mutagenesis using variousmutagens is often used. The surviving spores give rise to individualcolonies of isolates, whose capability of secondary metabolitesproduction is then tested. Mutants that exhibit poor growth andsporulation ability are not suitable candidates for furtherimprovement, even if their secondary metabolites production mayexceed that of the original strain. Todays high production strains,that synthesize as high as 10 000-fold levels of secondarymetabolites, compared to the original strains, are the result of manyyear of costly strain improvement. Unfortunately, these high

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production strains can revert to lose their overproduction thoughspontaneous mutagenesis.

When high production strains are prepared by mutagenesis, a type

of mutant that loses some of the structural genes can also beobtained. Such a mutant can exhibit a higher level of a secondarymetabolite intermediate whose transformation stopped due to theabsence of the corresponding enzyme. By crossing these mutants,some biosynthetic pathways used to synthesize secondarymetabolites were elucidated, e.g. tetracyclines (McCormick etall.1960).

Loss of the capability of secondary metabolite production in thestrains where extrachromosomal DNA was removed (e.g. by usingacriflavine or ethidium bromide) suggests that the regulatory genesare located on plasmids (Hotta et al, 1977; Okanishi, 1979; Akagavaet al., 1979; Boronin et al., 1974; Ikeda et al., 1982).

B. GENETIC MANIPULATION OF SECONDARY METABOLITESPRODUCERS

Structural genes for a number of secondary metabolites have beencloned into host microorganisms. Similarly, genes for secondarymetabolites resistance and other regulatory genes have also beencloned. Streptomyces lividans was found to be a suitable acceptor offoreign genetic material, in which a low degree of restriction of this

genetic material exists. This microorganism can host variousplasmids and phage vectors. However, at the same time, thismicroorganism was found not to be usable for the synthesis ofvarious secondary metabolites or of their high levels. The secondarymetabolites biosynthesis is a very complex process that requires notonly the structural genes for ESM but also the genes for regulation oftheir biosynthesis. Moreover, the overproduction of a secondarymetabolite has to be coordinated with the primary metabolism of theproducing microorganism.

The cloning of structural genes and genes for resistance to the ownsecondary metabolite enables us to work out genetic maps of theproducers. On the basis of those maps, hybrid clusters combined oftwo and more clusters of different secondary metabolites can becreated. Consequently, semisynthetic secondary metabolites can beproduced that may possess new biological activities or an antibioticactivity against resistant strains.

Polyketide synthase genes of microorganisms producing variouspolyketides have also been hybridized (Hopwood and Sherman,1990). As a result, a great similarity of polyketide synthases fromvarious streptomycetes was evidenced (Malpartida et al., 1987;Butler et al., 1990).

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IV. Obtaining new bioactive secondary metabolites

A. ISOLATION FROM NATURAL RESOURCES

In spite of the fact that several thousands of compounds isolatedfrom microorganisms having some biological activity are known, newsubstances are still saught by pharmaceutical companies. Theprobability of finding a new compound that would be usable as a newantibiotic or another biologically active compound is low, so a greatnumber of microorganisms have to be screened. A rough estimationsays that about 100 000 microorganisms are screened for thepresence of biologically active compounds per year. Modern screensare highly automated. The selection methods used, the targets, andthe methods of detection of the biological activity are normally notpublished.

Preparation of a new biologically active compound and itsintroduction into clinical practice requires the cooperation ofscientists 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 byStreptomyces and the rest mainly by moulds and non-filamentousbacteria. With an increasing spectrum of efficiency of microbialmetabolites, new, non-traditional sources of such compounds have

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been tappede. These include the microorganisms living underextreme conditions (high and low temperatures, etc.), sea livingmicroorganisms, and multicelular plants and animals. Another

important source of new compounds are the mutants of producers ofknown 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 tovirtually all fields of therapeutic interest, has proven successful andwill continue as an important avenue to new drug discovery. Theoriginal method for determination of antibiotic efficiency consisted ofthe application of test extract to wells made in agar medium layer in

Petri dishes to which the sensitive (target) microorganism wasinoculated. Most often Staphylococcus aureus, Sarcina lutea,Klebsiella pneumoniae, Salmonella gallinarium, Pseudomonas spp.,Bacillus subtilis, and Candida albicans were used. In case acompound with an antibiotic activity towards the testingmicroorganism was put into the well, it diffused through the agarmedium and a halo was formed around the well, as a result of thesuppressed growth of the microorganism. This classic plate assay hasbeen modified and improved in many ways.

The tests of other biological activities require different and

frequently sophisticated methods. This is true especially whenenzyme inhibitors are a case in point. Thus, Ogawara et al. (1986)chose a tyrosine protein kinase associated with the malignanttransformation of the cell caused by retroviruses as the target in abiochemical screen, they found genistein, an isoflavone fromPseudomonas, exhibiting a specific inhibitory activity. Production oftarget enzymes using recombinant DNA methodology hasdramatically expanded the number of potential targets that can befeasibly screened. A screen for the inhibitors of HIV reversetranscriptase is an example. The enzyme was produced inEscherichia 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. Theunspecificity of the enzyme systems facilitates the synthesis ofcertain secondary metabolites though the addition ofselectedprecursors to the growth medium. Thus, the reactionequilibrium can be shifted to promote the production of the derivative

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required, e.g. the prepareation of penicillins with different sidechains. The individual derivatives of penicillin and cephalosporin haveslightly different antimicrobial spectra and are active against

microorganisms resistant to other derivatives. The structuure ofpolypeptide antibiotics can also be modified by the addition of aminoacids to the growth

Replacement of a part of the metabolite molecule can beaccomplished chemically or enzymatically. In this way, semisyntheticpenicillins, cephalosporins, tetracyclines and other antibiotics can beprepared. The production of semisynthetic penicillins andcephalosporins is facilitated by the fact that 6-amino penicillanic and7-amino cephalosporanic acids are easily prepared.

The side chain is removed by the action of an enzyme or by achemical 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 andsomewhat different antimicrobial spectrum, as compared to theoriginal tetracyclines.

New derivatives of aminoglycosides also have been obtained bychemical and enzymatic modifications.

As the majority of bioactive products have rather complexstructures, their chemical synthesis is mostly more expensive than

the production by fermentation. An exception to the rule seems to bechloramphenicol, that is normally prepared using a chemicalsynthesis.

E. HYBRID BIOACTIVE PRODUCTS

Genetic engineering methods have recently advanced so muchthat now we can suitably combine structural genes of two or evenmore bioactive secondary metabolites producers. If these genes are

expressed, a hybrid bioactive products is synthesized, ore thatcannot be found in nature (Hutchinson, 1987, 1988; Tomich, 1988;Hopwood, 1993). Hopwood et al. (1985, 1986,) used this method withthe genes of actinorhodin synthesis and obtained related hybridmacrolides, mederhodin A and B, dihydromederhodin A anddihydrogranatirhodin. A new anthracyclines were produced when aDNA segment was cloned from Streptomyces purpurascenc ATCC25489 close to a region that hybridized to a probe containing part ofthe actinorhodin polyketide synthase Streptomyces galilaeus ATCC31615 (Niemi et al., 1994).

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V. Regulation of secondary metabolites production

A. GROWTH PHASES OF Stepromyces

In the cultures of Streptomyces capable of secondary metaboliteproduction several growth phases representing different physiologicalstatescan be distinguished:

1. Preparatory phase (lag phase) - the biomass increase is low, theculture is adapting to the new environment.

2. Growth phase (the term logarithmic phase is not suitable for mostStreptomyces since their growth curves are not exponentialfunctions) - intensive growth is taking place, accompanied by alow secondary metabolite synthesis. This phase is roughlyequivalent to "trophophase".

3. Transition phase - characterized by a decreased growth rate; thesecondary metabolite production is started. The enzymes ofsecondary metabolism are synthesized (B hal, 1986a; B hal, 1986b) and proteosynthesis slowed down.

4. Production phase - characterized by a significant reduction of thegrowth rate (sometimes growth is even completely ceased), anegligible change in the biomass concentration, and an intensivesynthesis of the secondary metabolite. This phase is some times

called "idiophase".

Producers of secondary metabolites mostly belong filamentousbacteria or fungi, which means that in their culture cells of variousage and at different stages of development are present. Themicroorganisms grow in pellets, inside which the cultivationconditions differ from those on the pellet surface (nutrientconcentrations, oxygen concentration, etc.). An increase in dryweight does always correlation with an increase growth since, instreptomycetes, often a thickening of the cell wall or glycocalyxformation occur that increase the dry weight value without rising the

number of cell (Vo ek et al., 1983). Since individual cells of a fermentation can be at different stages of development, (i.e. indifferent physiological states). The physiological state of the wholeculture represents an average of physiological states of the individualcells.

B. CONTROL OF FERMENTATION BY BASAL NUTRIENTS

In order to reach a high yieldof secondary metabolite, sufficientbiomass is required. Moreover to danger of contamination isdiminished and the economic parameters of the fermentation device

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are optimal if the growth is rapid. For this purpose, readily utilizablesources 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 nutrientsbecome limited.Thefore, the culture medium should be designed insuch a way that after the biomass increased sufficiently, at least oneof the nutrient sources will become depleted. Carbon source,nitrogen source and phosphate limitation have been described asimportant 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 theproducing microorganism and, after a time interval, another dose ofnutrients is added to the fermenter. Thus a prolonged cultivation canbe accomplished that enables us to increase the yield of the

secondary metabolite. The inflow of nutrients makes possible keeptheir optimal levels. An example of how a production cultivation ofStreptomyces aureofaciens can look like is shown in Fig. 21 (B hal, 1987). In cultivations whose course is well known, the nutrient inflowis programmed in advance

The inhibition of penicillin synthesis by glucose was observedshortly after its discovery in media containing glucose and lactose(Demain, 1974). The antibiotic was found to be synthesized only afterglucose was depleted from the medium and lactose started to be

metabolized. Similarly, glycerin was observed to inhibit thebiosynthesis of cephalosporins (Demain, 1983). Using these data,fermentation protocols were worked out, in which the level of glucosewas kept low so as not to inhibit the antibiotic production. Themechanism of inhibition of the secondary metabolites synthesis byreadily utilizable sugars probably consists in a repression of enzymesof secondary metabolism (Revilla, G. et al., 1986; Erban, et al., 1983).

Readily utilizable nitrogen sources can also negatively influencethe production of secondary metabolites.Ammonium ions oftendecrease secondary metabolite synthesis and, therefore, theirconcentration in production media is limited while, soy flour, peanut

flour and other substances are preffered nitrogen sources. Theselatter nitrogen sources are more similar to those used by themicroorganisms producing secondary metabolites in nature. Readilyutilizable nitrogen sources repress enzymes of secondary metabolismin Cephalosporium acremonium (Shen et al., 1984) during thebiosynthesis of cephalosporin and in Streptomyces clavuligerusproducing cephamycin (Demain and Brana, 1986). Similarly, theinhibition of biosyntheses of leucomycin (Omura et al., 1980a), tylosin(Omura et al., 1980b), and erythromycin (Flores and Snches, 1985)are explained by the repression of enzymes of secondarymetabolism. Ammonium salts also inhibit the activity of

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anhydrotetracycline oxygenase isolated from S. aureofaciens (B hal et al., 1983). The overproduction of most secondary metabolites can be

achieved only if phosphate is limited. Inorganic phosphate has to becarefully added in doses to the medium so as to accomplish anoptimal ratio between biomass production and secondary metabolitebiosynthesis. When bound to organic compounds normally added tomedium (soy flour, etc.), phosphate does not affect secondarymetabolite production. In general secondary metabolite biosynthesisis started when the concentration of phosphate decreased below acertain level. At this point, the producer culture undergoes a shiftfrom the physiological state characteristic for the growth phase tothat of the overproduction phase.

Inorganic phosphate also causes a repression of the synthesis of

enzymes of secondary metabolism (B hal et al., 1979b; Madry and Pape; 1981, Martin et al., 1981). After phosphate was depleted fromthe medium, a significant decrease of the rate of proteosynthesis wasobserved during tetracycline (B hal,1982). If phosphate was kept above the threshold concentration, the significant decrease of therate of protein synthesis did not occur and ESM were not synthesized.An addition of phosphate to the medium at the beginning of theproduction phase, after the phosphorus source was depleted and theenzymes of secondary metabolism synthesis initiated, resulted in adecrease 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 theinitiation of the secondary metabolite synthesis does not significantlydiffer from the transduction of signals for other metabolic processes.Catabolite repression signals or those signalling the depletion ofnitrogen or phosphate, or the initiation of sporulation, aretransducted via two-component signal proteins ( Doull and Vining,1995). With some structural varietion, these proteins are

characterized by common mechanistic features and conserved aminoacid 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-transmitteris composed of asensordomain located near its N-end; the N-end is found outside thecytoplasm. A specific effector is capable of binding directly to this N-end. The transmitterdomain is located in the cytoplasm to be linkedto the sensor domain via a hydrophobic, amino acid sequencestretching across the membrane. The sensor-transmitterproteins arehistidine-protein kinases, capable of autophosphorylation at their C-

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ends on receiving a proper signal. The phosphorylated proteinbecomes a donor in reactions transferring phosphorus. The acceptoris the cytoplasmic, response-regulator protein. Two-component

signal proteins thus transfer the information concerning theconditions that can affect the cell action.

D. REGULATION BY LOW MOLECULAR COMPOUNDS

The expression of structural genes is also regulated by some lowmolecular compounds. The mechanism of their action is notunderstood. For example tryptophan exhibited a stimulatory effect onthe production of mucidin in the basidiomycete Oudemansiellamucida (Nerud et al., 1984) and actinomycin in Streptomycesparvulus (Troast et al., 1980). Methionine was found to promote thesynthesis of cephalosporin C (Nuesch et al., 1973). Neithertryptophan nor methionine were used as building units for thesemetabolites.

Benzyl thiocyanate is one of the low molecular compounds thataffect the chlortetracycline biosynthesis. It increases the productionof both chlortetracycline and tetracycline in S. aureofaciens,although, it does not influence the production of oxytetracycline in S.rimosus. The effect on the metabolism ofS. aureofaciens is multiple(Novotn et al., 1995), including a number of enzymes, including the

enzymes of secondary metabolism (B hal et al., 1982). Benzyl thiocyanate is able to raise the level of secondary metaboliteproduction only if it is added in the lag phase, growth phase or at thebeginning of the production phase. Its effect is more pronounced inlow production strains, where the enzyme level and chlortetracyclineproduction are increased 10 to 20-fold, as compared to highproduction 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 mostfamous of them is factor A, -butyrolactone (Fig. 22), that was discovered in Streptomyces griseus (Khokhlov et al., 1969; Khokhlov,1982). A non-producing strain started the synthesis of streptomycinafter factor A was added to the culture simultaneously, the colturaformed aerial mycelium. Factor A is synthesized by manystreptomycetes but the regulatory effect was observed only inStreptomyces griseus, Streptomyces bikiniensis and Streptomycesactuosus (Ohkishi et al., 1988). The addition of factor A to blockedmutants ofStreptomyces griseusJA 5142, caused resumption of the

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synthesis of anthracyclines and leukaemomycin (anthracycline typeantibiotic) (Graefe et al., 1983). The resistance to streptomycin linkedwith 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 -butyrolactones. Virginiae butanolides were detected in Streptomycesvirginiae (Yanagimoto et al., 1979). Factor I was isolated fromStreptomyces sp. FR1-5 (Sato et al., 1989) and its effectiveconcentration 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 ina blocked mutant ofNocardia 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. Thestructure of factor B is similar to cAMP but none of the derivatives ofknown nucleotides exhibited a comparable effect. Chemicallyprepared derivatives of factor B have also been tested. Activity wasobserved with those that contained a C2 -C12 acyl moiety; octylesterwas the most effective of them (Kawaguchi et al., 1988). Asubstitution of guanosine for adenine did not result in a loss of thebiological activity of factor B.

Factor C was isolated from the fermentation medium ofStreptomyces griseus.This compound causes cytodifferentiation of

non-differentiating mutants (Szabo et al., 1967). Factor C is a proteinhaving a molecular weightof about 34 500 D, and is rich inhydrophobic amino acids.

The effect of autoregulators is easily observable if they elicitmorphological changes such as the formation of aerial mycelium.Carbazomycinal and6-methoxcarbazomycinal, isolated from Streptoverticillium species,inhibit of the aerial mycelium formation at a concentration of 0.5 to 1microgram per ml. Autoregulators affecting sporulation were found inStreptomyces venezuelae (Scribner et al., 1973), Streptomycesavermitilis (Novk et al., 1992), and Streptomyces

viridochromogenes NRRL B-1551 (Hirsch and Ensign, 1978). From thesame strain ofStreptomyces viridochromogenes, germicidin wasisolated by Petersen and coworkers (1993). The compound had aninhibitory effect on the germination of arthrospores ofStreptomycesviridochromogenes at a concentration as low as 40 picogram per ml.Germicidin (6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone) is the first knownautoregulative inhibitor of spore germination in the genusStreptomyces and was isolated from the supernatant of germinatedspores and also from the supernatant of a submerged culture.

Mutants ofStreptomyces cinnamonensis resistant to highconcentrations of butyrate and isobutyrate produce an anti-

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isobutyrate (AIB) factor that is excreted into the culture medium(Pospil, 1991). On plates, AIB factor efficiently counteracted toxicconcentrations of isobutyrate, acetate, propionate, butyrate, 2-

methylbutyrate, valerate, and isovalerate in Streptomycescinnamonensis and other Streptomyces species.

F. REGULATION BY PHOSPHORYLATED NUCLEOTIDES

Global control mechanisms for secondary metabolites biosynthesishave been investigated. The energetic state of the cell is thought tobe such a general control mechanism. The intracellular ATP levelreflects the content of free energy in the cell. In some cases, the startof the secondary metabolite synthesis is linked with a decrease of theintracellular ATP level. Such a relationship was observed inStreptomyces aureofaciens and Streptomyces fradiae during theproduction of tetracycline (Janglov et al., 1969; urdov 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 isaccompanies active primary metabolism. A slow down of growth andprimary metabolism is accompanied by a decrease of the ATP level.

The role of cAMP in the metabolism of secondary metabolitesproducers was also studied, especially in connection with glucoseregulation. Hitherto, no indication has been obtained suggesting a

specific role of cAMP in the regulation of secondary metabolitesproduction (Cortz et al., 1986; Chatterjee and Vining, 1981).

G. REGULATION BY METAL IONS

Metal ions act as a part of enzyme active centers. The optimalconcentrations of metal ions for cultivation of the secondarymetabolites producing strains have usually been determinedempirically. In complex media it is generally not necessary to addspecific metal ions, however in defined media their presence isessential.

VI. Resistance to bioactive products

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

A. RESISTANCE OF SECONDARY METABOLITES PRODUCERS

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Basic metabolic processes of wild type, secondary metaboliteproducing microorganisms are not inhibited if the secondary

metabolires are synthesized at low concentrations. After strainimprovement, strains with 100 to 1000-fold increases insecondarymetabolite yields have been isolated. Genome changes of theimproved strains include a number of deletions and amplifications inthe 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 theenzyme activities that participate in the synthesis of the secondarymetabolite. In high production strains, such controls are lost and thestrains 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 expressedsimultaneously with the structural genes. The genes of newly gainedresistances, 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 itand then resistance comes into existence. It is not known whether adecreased ability to bind the secondary metabolites results from aposttranslational modification of the active centre or if resistant

molecules of the enzyme are synthesized de novo. Clear evidence insupport 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 elongationfactors are also impaired, together with reduced synthesis ofguanosine penta- and tetraphosphates (Weiser et al., 1981). Theantibiotic producers (mostly Streptomyces), as well as the bacteriaagainst which the antibiotic is used, protect themselves byposttranscriptional modification of rRNA. Adenine is methylated toobtain N6-dimethyladenine rRNA in the 23S subunit. Such modifiedribosomes do not bind the antibiotic. In other cases, adenine is

methylated to yield 2-O-methyladenosine (Cundliffe and Thompson,1979; Mikulk et al.,1983; Thompson et al., 1982). However,methylation modified ribosomes can be sensitive to the effect ofother antibiotics. The genes coding for methylases, that catalyzemethylation of adenine in some Streptomycetes, were cloned intoStreptomyces lividans and the ribosomes of the mutants preparedwere resistant towards the corresponding antibiotics.

The most important mechanism of resistance observed in thesecondary metabolites producers seems to be export from the cell tothe environment. In Streptomyces rimosus, an oxytetracyclineproducer, genes for the enzymes increasing the antibiotic transport

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rate precede the structural genes on the chromosome. Genes for theresistance consisting in the protection of ribosomes via the synthesisof 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 theproblem of a reverse flow of products into the cell. Some secondarymetabolites are bind to the cell wall, others are complexed in themedium (tetracyclines in the presence of Ca2+ ions). Cytoplasmicmembranes of resistant strains are often less sensitive to the effectof secondary metabolites. This kind of resistance is thought to beconnected with the content of phospholipids in the cell.

Secondary metabolite producers can use several types ofresistance simultaneously. Tetracyclines, that strongly inhibit proteinsynthesis, interfere with the binding of the ternary complex of amino

acyl-tRNA-EFTu-GTP to ribosomes (Gavrilova et al., 1976). The genesfor resistance were cloned into Streptomyces griseus, sensitive totetracyclines, using pOA15 as the vector plasmid (Ohnuki et al.,1985). After mapping the plasmids in resistant strains usingrestriction nucleases, two types of plasmids capable of transfer ofdifferent types of resistance were found. One type consisted in anincreased ability of tetracycline transport to the medium, the other inan increased resistance of ribosomes to the effect of tetracyclines.These ribosomes bore a compound(s), bound to their surface, thatcould be removed by washing with 1 M NH4Cl solution. The ribosomes

lost their resistance after the washing, which was demonstrated withboth the ribosomes ofStreptomyces griseus and those of the originalstrain ofStreptomyces rimosus.The two types of resistance wereboth constitutive and inducible. The inhibiting concentrations ofchlortetracycline in Streptomyces aureofaciens are higherin theproduction phase as compared to the growth phase (B hal et al., 1979a). Thus, the resistance can be increased even during thefermentation process.

Another way secondnary metabolite producers can avoid the effectof their products is to situate the distal enzymes of secondarymetabolite biosynthetic pathway (synthases) outside the cell, most

often in the periplasm. In Streptomyces aureofaciens, a higherproportion of the terminal enzyme of tetracycline synthase was foundunder high production conditions in periplasm, as compared to lowproduction conditions (Erban et al.,1985).

B. RESISTANCE IN PATHOGENIC MICROORGANISMS

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Shortly after antibiotics were introduced into clinical practice on amassive scale, strains of hitherto-sensitive microorganisms started toappear. These resistant strains required the use of much higher

antibiotic concentrations or, were completely resistant to theseantibiotics. The resistant strains originated from clones that survivedthe antibiotic treatment, especially if the treatment was terminatedbefore all pathogenic microorganisms were killed or the antibioticwas applied at sublethal doses.

There are several ways in which microorganisms can gainresistance (Ogawara, 1981). These include:1. Creation of an alternative metabolic pathway producing acompound whose biosynthesis is blocked by the bioactive metabolite;2. Production of a metabolite that can antagonize the inhibitoryeffects of the bioactive metabolite; 3. Increase of the amount of the

enzyme inhibited by the secondary metabolite; 4. Decrease of thecells metabolic requirement for the reaction inhibited by thesecondary metabolite; 5. Detoxification or inactivation of thebioactive metabolite; 6. Change of the target site; 7. Blocking of thetransport of the bioactive metabolite into the cell.

In most resistant microorganisms, the mechanisms of resistancementioned in the items 5, 6 and 7 are encountered.

Penicillins and cephalosporins are degraded using three ways: bythe enzyme penicillin amidase that cleaves the amidic bond by whichthe side chain is bound to the -lactam ring; by the enzyme acetyl

esterase that hydrolyzes the acetyl group at C-3 on the dihydrazinering of cephalosporins and by the enzyme -lactamase that catalyzes hydrolysis of the -lactam ring of penicillins and cephalosporins.

Penicillin amidases are rarely used by microorganisms to build upresistance to -lactam antibiotics, however these enzymes are often employed for the synthesis of semisynthetic antibiotics. Acetylesterase is also not important from the point of view of antibioticresistance. In most cases, -lactams are inactivated by -lactamase that destroys one of the important sites for their antibiotic activity;the damage is irreversible.

-lactamases, however, are not only synthesized by

microorganisms that came into contact with penicillins. Constitutivesynthesis of these enzymes have been found in three quarters of allstreptomyces strains, (Ogawara et al., 1978). One can suppose thatthe genes for the synthesis of -lactamases were transferred horizontally. Recent studies indicate frequent and promiscuous genetransfer even between distantly related bacterial species. Apossibility of direct transfer from a streptomycete to a pseudomonad,for example, may seem unlikely. However, it is not necessary toinvoke direct exchanges. It is more reasonable to imagine thatdistant exchanges between distantly related organisms result from acascade of transfer between related species (Davis, 1992).

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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 isliberated that inhibits glutamine synthetases, key enzymes of theinorganic nitrogen assimilation pathway.

VIII. References

Akagava, H., Okanishi, M., and Umezava, H. (1979). Genetics andbiochemical studies of chloramphenicol nonproducing mutants ofStreptomyces venezuelae carrying plasmid.J. Antibiot.32, 610-620.

Bayer, H., Gungel, K. H., Hagele, K., Hagenmayer, H., Jessipow, S.,Koenig, W. A., and Zaehner, H. (1972). Stoffwechselproducte vonMicroorganismen. Helv. Chim. Acta55, 224-239.

B hal, V., Van k, Z., Ho lek, Z., and Ramadan, A. (1979a). Synthesis and degradation of proteins and DNA in Streptomycesaureofaciens. Folia Microbiol. 24, 211-215.

B hal, V., Ho lek, Z., and Van k, Z. (1979b). Anhydrotetracycline oxygenase activity and biosynthesis of tetracyclines in Streptomycesaureofaciens. Biotechnol Lett.1, 177-182.

B hal, V. (1982). Oligoketide-synthesizing enzymes. In:

"Overproduction of Micobial Products" (V. Krumphanzl, B. Sikyta., Z.Van k and D. W. Tempest, Eds.), pp. 301-309. Academic Press, London.

B hal, V., Buko, M., and Ho lek, Z. (1983). Tetracyclines. In:"Biochemistry and Genetic Regulation of Comercially ImportantAntibiotics". ( L. C. Vining, Ed.) pp. 255-276. Addison-Wesley Pub.Comp., London.

B hal, V., Neuil, J., and Ho lek, Z. (1983). Effect of tetracycline derivations and some cationts on the activity of anhydrotetracyclineoxygenase. Biotechnol. Lett. 5, 537-542.

B hal, V. (1986a). Enzymes of secondary metabolism in microorganisms. Trends Biochem. Sci.11, 88-91.

B hal, V. (1996b). Enzymes of secondary metabolism: regulation of their expression and activity. In: "Regulation of Secondary MetaboliteFormation" (H. Kleinkauf, H. von Doehren., H. Dornauer and G.Nasemann., Eds.), pp. 269-281. VCH Verlagsgesselshaft, Weinheim.

B hal, V. (1987). Tetracycline fermentation at its regulation. CRCCrittical Reviews in Biotechnology 5, 275-318.

24

24


25/67

B hal, V., and Hunter, I. S. (1995). Tetracyclines. In: "Genetics andBiochemistry of Antibiotics Production"(L. C .Vining and C. Stuttard,Eds.), pp. 359-384. Butterworth-Heinemann, Boston.

Bennett, J.W. and Bentley R. (1989). What is a name?-Microbialsecondary metabolites.Adv. Appl. Microbiol. 35,1-28.

Bentley, R., and Bennett, J.W. (1999). Constructinc polyketides: FromCollie to combinatorial biosynthesis.Ann. Rev. Microbiol. 53, 411-446.

Billich, A., and Zocher, R. (1987). Enzymatic synthesie of cyclosporineA.J. Biol. Chem. 262, 17258-17259.

Binnie, B., Warren, M., and Butler, M. J. (1989). Cloning andheterologous expression in Streptomyces lividans ofStreptomycesrimosus genes involved in oxytetracycline biosynthesis. J. Bacteriol.

171, 887-895.Birch, A. W., and Robinson, J. A. (1995). Polyethers. In: "Genetics andBiochemistry of Antibiotics Production"(L. C .Vining and C. Stuttard,Eds.), pp. 443-476. Butterworth-Heinemann, Boston.

BuLock, J. D. (1961). Intermediary metabolism and antibioticsynthesis.Adv. Appl. Microbiol. 3, 293.

BuLock J.D., and Ryan, A. J. (1958). The biosynthesis of patulin. Proc.Chem. Soc. 222-223

Burg, R.W., Miller, B.M., Baker, E.E, and al. (1979). Avermectins, new

family of potent anthelmintic agents: Production organism andfermentation. Antimicrob. Agents Chemother. 15, 361-367.

Campeneere, D. D., Baourain, R., Huybrechts, M., and Trouet, A.(1979). Comparative study in mice of the toxicity, pharmacology, andtherapeutic activity of daunorubicin-DNA and doxorubicin-DNAcomplex. Chem. Pharm. Bull. 37, 1639-1641.

Corcoran, J. W., and Chick, M. (1966). Biochemisry of the macrolideantibiotics. In: "Biosynthesis of Antibiotics" (J. F. Snell, Ed.), pp.149-201. Academic Press, New York.

Cundliffe, E., and Thompson, J. (1979). Ribosome methylation andrezistance to thiostrepton. Nature, 278, 859-861.

Davis, J. (1992). Another look at antibiotic rezistance.J. Gen.Microbiol. 138, 1553-1559.

Demain, A. L. (1974). Biochemistry of penicillin and cephalosporinfermentation. Lloydia37, 147-167.

Demain, A. L. (1983). Biosynthesis of -lactam antibiotics. In:"Handbook of Experimental Pharmacology" (A. L. Demain and N. ASolomon., Eds.), Vol. 67, pp.189-228. Springer Verlag.

25

25


26/67

Demain, A. L., and Bra a, A. F. (1986). Control of cephalosporin formation in Streptomyces clavuligeerus by nitrogen compounds. In:"Regulation of Secondary Metabolite Formation"(H. Kleinkauf, H. von

Doehren, H. Dornauer and G. Nasemann, Eds.), pp. 77-88. VCHVerlagsgesselshaft, Weinheim.

Dimroth, P., Walter, H., and Lynen, F. (1970). Biosynthesis von 6-Methylsalicylisaure. Eur. J. Biochem. 13, 98-110

Dimroth, P., Ringelmann, E., Lynen, F. (1976). 6-Methylsalicylic acidfrom Penicillium patulum. Eur. J. Biochem. 68, 591-596.

Doull, J. L., and Vining, L. C. (1995). Global physiological controls. In:"Genetics and Biochemistry of Antibiotics Production" (L. C. Viningand C. Stuttard, Eds.), pp. 9-63. Butterworth-Heinemann, Boston.

Erban, V., Novotn, J., B hal, V., and Ho lek, Z. (1983) Growth rate, sugar consumption and the expression of anhydrotetracyclineoxygenase in Streptomyces aureofaciens. Folia Microbiol. 28, 262-267.

Erban, V., B hal, V., Trilisenko, L., Neuil J., and Ho lek, Z. (1985). Tetracycline dehydrogenase: spectroscopic assay, propeties andlocalization in strains ofStreptomyces aureofaciens. J. Appl. Biochem.7, 341-346.

Flores, E., and Sanches, S. (1985). Nitrogen regulation oferythromycin formation in Streptomyces erythreus. FEMS Microbiol.Lett. 26, 191-194.Gavrilova, L.P., Kostiashima, O., Koreliansky, V.E., Rutkevitch, N.M.,Spirin, A.S. (1976). Factor free (non-enzymatic) and factor dependentsystem of translation of polyuridylic acid by Escherichia coliribosomes.J. Mol. Biol. 101, 537-542.

Goldman, P., and Vagelos, P.R. (1962). The formation of enzyme-bound acetoacetate and its conversion to long chain fatty acids.Biochem. Biophys. Res. Comm. 7, 414-418.

Graefe, U., Schade, W., Eritt, I., and Fleck, W. F. (1982). A new inducer

of anthracycline biosynthesis from Streptomyces viridochromogenes.J. Antibiot. 35, 1722-1723.

Hara, O., and Beppu, T. (1982). Induction of streptomycin-inactivatingenzyme by A-factor in Streptomyces griseus. J. Antibiot. 35, 1208-1215.

Harris, D. R., McGeachin, S. G., and Mills, H.H. (1965). The structureand stereochemistry of erythromycin A. Tetrahedron Lett. 679-685.

Harris, C. M., and Harris, T. M. (1982). Structure of the glycopeptideantibiotic vancomycin. Evidence for an asparagine residue in thepeptide.J. Amer. Chem. Soc. 104, 4293-4295.

26

26


27/67

Hirsch, C. F., and Ensign, J. C. (1978). Some properties ofStreptomyces viridochromogenes spores.J. Bacteriol. 134, 1056-1063.

Hopwood, D. (1993). Genetic enginering ofStreptomyces to createhybrid antibiotics. Curr-Opin. Biotechnol. 4, 531-537.

Hopwood, D. A., Malpartida, F., Kieser, H. M., Ikeda, H., and Duncan, J.(1985). Production of "hybrid" antibiotics by genetic engineering.Nature314, 624-644.

Hopwood, D. A., Malpartida, F., and Chater, K. F. (1986). In:"Regulation of Secondary metabolite Formation"(H. Kleinkauf, H. vonDoehren, H. Dornauer and G. Nasemann, Eds ), pp. 23-33. VCHVerlagsgesselshaft, Weinheim.

Hopwood, D. A., and Sherman, D. H. (1990). Molecular genetic ofpolyketides and its comparison to fatty acid biosynthesis.Ann. Rev.Genet. 14, 37-66.

Horinuchi, S., and Beppu, T. (1990). Autoregulatory factors ofsecondary metabolism and morphogenesis in actinomycetes. Crit.Rev. Biotechnol. 10, 191-204.

Horinuchi, S., and Beppu, T. (1992). Autoregulatory factors andcomunicatio in actinomycetes.Ann. Rev. Microbiol. 46, 377-398.

Horinouchi, S., and Beppu, T. (1995). Autoregulators. In: "Genetics

and Biochemistry of Antibiotics Production"(L. C .Vining and C.Stuttard, Eds.), pp. 103-119. Butterworth-Heinemann, Boston.

Hotta, K., Okami, Y., Umezawa, H., Huang, M., and Gipson, F. ( 1977).Elimination of the ability of kanamycin-producing strain tobiosynthesis deoxystreptamine moiety by acriflavine.J. Antibiot. 30,1146-1149.

Hutchinson, C. R. (1987). The inpact of genetic engineering on thecommercial production of antibiotics by Streptomyces and relatedbacteria.Appl. Biochem. Biophys.16, 169-190.

Hutchinson, C. R. (1988). Prospects for the discovery of new (hybrid)

antibiotics by genetic engineering of antibiotic-producing bacteria.Medicinal Res. Rev. 8, 558-567. Hutchinson, C. R. (1995).Anthracyclines. In: "Genetics and Biochemistry of AntibioticsProduction" (L. C. Vining and C. Stuttard, Eds.), pp. 331-357.Butterworth-Heinemann, Boston..Iitaka, Y. (1978). Molecular conformations of bioactive peptides incrystals. In: "Bioactive Peptides by Microorganisms" (H. Umezava, T.Takita and T. Shiba, Eds.), 153-182. Kadansha, Tokyo.

Ikeda, H., Tanaka, H., and Omura, S. (1982). Isolation andcharacterization of covalently closed circular DNA associated with

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27


28/67

chromosomal and membrane fraction from Streptomycesambofaciens.J. Antibiot. 35, 497-516.

Ikeda, H., and Omura, S. (1995). Control of avermectin biosynthesis inStreptomyces avermectilis for the selective production of usefulcomponent.J. Antibiot. 48, 549-562.

Ishihara, H. M., Hara, N., and Iwabuchi, T. (1989). Molecular cloningand expression in Escherichia coli ofBacillus licheniformis bacitracinsynthetase gene 2 gene. J. Bacteriol.171, 1705-1711.

Janglov, Z., Such, J., and Van k, Z. (1969). Regulation of biosynthesis of secondary metabolites. VII. Intracellular adenosin-5-triphosphate concentration in Streptomyces aureofaciens. FoliaMicrobiol. 14, 208-210.

Jensen, S. E., and Demain A. L., (1995). Beta-Lactams. In: "Geneticsand Biochemistry of Antibiotics Production" (L. C. Vining and C.Stuttard, Eds.), pp. 239-268. Butterworth-Heinemann, Boston..

Kawagushi, T., Asahi, T., Satoh, T., Uezumi, T., and Beppu, T. (1984).B-factor an essential regulatory substance inducing the production ofrifamycin in a Nocardia sp.J. Antibiot. 37, 1587-1595.

Khokhlov, A. S. (1982). Low molecular weight microbial bioregulatorsof secondary metabolites. In: "Overproduction of Micobial Products"( V. Krumphanzl, B. Sikyta, Z. Van k and W. D. Tempest, Eds.), pp. 97-109. Academic Press, London.

Kleinkauf, H., von Doehren, H. In: "Regulation of SecondaryMetabolite Formation"(H. Kleinkauf, H. von Doehren, H. Dornauerand G. Nasemann, Eds.), pp. 173-207. VCH Verlagsgesselshaft,Weinheim.

Kleinkauf, H., von Doehren, H. In: "Biochemistry and GeneticRegulation of Commercially Important Antibiotics" (L. C. Vining, Ed.)pp.95-145. Addison-Wesley Publishing Company, London.

Laland, S. G., and Zimmer, T-L. (1973). Bioactive peptides producedby microorganisms. Essays Biochem. 9, 31-57.

Lipman, F., (1971). Attempts to map a prcess evolution of peptidebiosynthesis. Science 173, 875-884.

Levi-Schaff, F., Bernstein, A., Meshore, A., and Arnon, R. (1982).Reduced toxicity of daunorubicin by conjugation to dextran. CancerTreat. Terp. 66, 107-114.

Liu, C. M., McDanie, L. E., and Schaffner, C. P. (1972). Studies oncandicidin biosynthesis.J. Antibiot. 25, 116-212.

Lotvin, J. A., Ryan, M. J., and Strahty, N. (1992). European PatentApplication 91110631,8.

28

28


29/67

Lynen, F. (1959). Participation of acyl-CoA in carbon chainbiosynthesis.J. Cell.

Comp.Physiol. 54, Supplement 1:33-49.

Lynen, F., and Reichert, E. (1951). Zur Chemischestructur der"Aktivierte Essigsaure".Angew. Chem. 63, 47-48.

Lynen, F., and Tada, M. (1961). Die biochemische Grundlage der"Polyacetate-Regel".Angew. Chem. 73, 513-519.

Madry, N., and Pape, H. (1981). Regulation of tylosin biosynthesis byphosphate - possible involvement of transcriptional control. In:"Actinomycetes" (K. P. Schall and G. Pulverer, Eds.), pp. 441-445. Zbl.Bact. Suppl., G. Fischer, Stutgart, New York.

Malpartida, F., Hallam, S. E., and Kieser, H. W. (1987). Homologybetween Streptomyces genes coding for synthesis of differentpolyketides used to clone antibiotic biosynthetic genes. Nature325,818-821.

Martin, J. F., and Liras, P. (1989). Beta-lactams.Adv. Biochem. Eng.39,153-187.

Martin, J. F. (1992). Clusters of genes for the biosynthesis ofantibiotcs: regulatory genes and overproduction of pharmaceuticals.J. Ind. Microbiol. 9, 73-90.

McCormick, J. R. D., Hirsch, U, Sjolander, N. O., and Doerschuk, A. P.

(1960). Cosynthesis of tetracyclines by pairs ofStreptomycesaureofaciens mutants.J. Am. Chem. Soc. 82, 5006-5009.

McCormick, J. R. D. (1965). Biosynthesis of tetracyclines. In:"Biosynthesis of Antibiotic Substances"(Z. Van k and Z. Ho lek, Eds.), pp. 73-79. Academic Press, Praha.McDaniel, R., Ebert-Khosla, S., Hopwood, D.A., and Khosla, C. (1993).Engineering biosynthesis of novell polyketides. Science 262,1546-1550.

MacNeil, D. J. (1995). Avermectins. In: "Genetics and Biochemistry ofAntibiotic Production" (L. C. Vining and C. Stuttard, Eds.), pp. 421-

442. Stuttard, Butterworth-Heinemann, Boston.Malpartida, F., and Hopwood, D. A. (1984). Molecular cloning of thewhole biosynthetic patway of a Streptomyces antibiotic and itsexpression in a herogenous host. Nature 309, 462-464.

Martin, J. F. (1977). Biosynthesis of polyene macrolide antibioics.Ann.Rev. Microbiol. 31, 13-38.

Martin, J. F., and Gil, J. A. (1979). Biosynthesis and attachment ofamminosugars to polyene macrolide antibiotics.J. Antibiot. 32, 5122-5128.

29

29


30/67

Martin, J. F., Alegre, M. T., Gil, J. A., and Naharro, G. (1981). Polyenesantibiotics. In: "Advances in Biotechnology: Fermentation Products"(C. Vezina and K. Singh, Eds.), Vol. III, pp.129-134. Pergamon press,

Toronto.Mikulk, K., Jir ov, A., Janda, I., and Weiser J. (1983). Susceptibility of ribosome af the tetracycline-producing strain ofStreptomycesaureofaciens to tetracycline. FEBS Lett. 152, 125-130.

Miller, P. A., Hash, J. H., Lincks, M., and Bohonos, N. (1965).Biosynthesis of 5-hydroxytetracycline. Biochem. Biophys. Res.Commun. 18, 325-331.

Miller, T. W., Chaiet, L., Cole, D. J., and al. (1979). Avermectins, newfamily of potent anthelminic agents: Isolation and chromatogrphic

properties.Antimicrob. Agents Chemother.15

, 368-371.Moore, C., and Pressman, B. C. (1964). Mechanism of action ofvalinomycin on mitochondrie. Biochem. Biophys. Res. Commun. 15,562-567.

Nerud, F., Zouchov, Z., and Muslek, V. (1984). Effect of tryptophanon ezymes of aromatic acids metabolism in Oudemansiella mucida.Folia Microbiol. 29, 389-402.

Niemi, J., Ylihoko, K, Hakala, J., Parssinen, R., Kopio, A., and Mansala,P. (1994). Hybride anthracycline antibiotics: production of newanthracyclines by cloned genes from Streptomyces purpurascens inStreptomyces galilaeus. Microbiology. 140, 1351-1358.Novk, J., Kopeck, J., and Van k, Z. (1992). Sporulation-inducing factor in Streptomyces avermitilis. Folia Microbiol. 37, 463-465.

Novotn, J., Erban, V., Pokorn, V., and Ho lek, Z. (1983). Benzylthiocyanate: An effector of development and chlortetracyclineproduction in Streptomyces aureofaciens. In Abstract Book of"Genetics and Differentiation of Actinomycetes", p. 69. Weimar.

Novotn, J., Li, X-M, Novotn, J. J., Vohradsk, J., and Weiser, J. (1995).Protein profiles ofStreptomyces aureofaciens producing

tetracyclines. Reappraisal of the effect of benzyl thiocyanate. CurrentMicrobiol. 31, 84-91.

Ogawara, H. (1981. Antibiotic rezistance in pothogenic and producingbacteria, with special reference to -lactam antibiotics. Microbiol Rev.45, 591-619.

Ogawara, H., Akiyama, T., Ishida, J., Watanabe, S., and Suzuki, K.(1986). A specific inhibitor for tyrosine protein kinase fromPseudomonas. J. Antibiot. 39, 606-608.

Okanishi, M. (1985). Function of plasmids in aureothricin production .Trend in Antibiot. Res. 23, 32-41.

30

30


31/67

Ohnuki, T., Katoh, T., Imanaka, T., and Aiba, S. (1985). Molecularcloning of tetracycline resistance genes from Streptomyces rimosusin Streptomyces griseus and characterization of the cloned genes.J.

Bacteriol. 161, 1010-1016.Omura, S., Nakagawa, A., Takeshima, H., Miyazava, J., and Kitao, C.(1975). A 13Cnuclear magnetic study of the biosynthesis the 16-membered macrolide antibiotic tylosin. Tetrahedron Lett. 4503-4506.

Omura, S., Tanaka, Y., Takahashi, Y., and Iwai, Y. (1980a).Stimulation of leucomycin production by magnesium phosphate andits relevance to nitrogen catabolite regulation.Antimicrob AgentsChemother. 18, 691-695.

Omura, S., Tanaka, Y., Takahashi, Y., and Iwai, Y. (1980b).

Stimulation of the production of antibiotics by magnesium phosphateand related insoluble materials. J. Antibiot. 33, 1568-1569.

Omura, S., Ikeda, H., Malpartida, F., Kieser, H. M., and Hopwood, D. H.(1986). Production of new hybrid antibiotics, mederrhodin-A, andmederrhodin B, by a genetically engeneering strain.Antimicrob.Agents Chemother. 29, 13-19.

Petersen, F., Zaehner, H., Metzger, J. W., Freund, S., and Hummel, R-P.(1993). Germicidin, an autoregulative germination inhibitor ofStreptomyce viridochromogenes NRRL B-1551. J. Antibiot. 46, 1126-1138.

Pospil, S. (1991). Rezistance ofStreptomyces cinnamonensis tobutyrate and isobutyrate: production and properties of new ant-isobutyrate (AIB) factor.J. General Microbiol. 127, 2141-2146.

Pospi, S., Sedmera P., Havrnek, M., Krumphanzl, V., and Van k, Z. (1983). Biosynthesis of monensins A and B.J.Antibiot.36, 617-619.

Reading, C., and Cole, M. (1977). Clavulanic acid.Antimicrob. AgentsChemother. 11, 852-857.

Revilla, G., Ramos, F. R., Lpez-Nieto, M. J., Alvarez, E., and Martin, J.F. (1986). Glucose represses formation of -(L- -aminoadipyl)- L-

cysteinyl-D-valine and isopenicilin A synthase but not penicillinacyltransferase in Penicillium chrysogenum. J. Bacteriol. 168, 947-952.

Robinson, J. A. (1991). Polyketide synthase complexes: their structureand function in antibiotic biosynthesis. Philos. Trans. R. Soc. Lond. B.Biol. Sci. 332, 107-114.

Roland, I., Froyshov, O., and Laland, G. (1977). A rapid method for thepreparation of three enzymes of bacitracin synthetase essentialy freefrom other proteins. FEBS Lett. 84, 22-24.

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31


32/67

Scribner, H. E., Tang, T., and Bradley, S. G. (1973). Production of asporulation pigment by Streptomyces venezuelae. Appl. Microbiol.25, 873-879.

Sekiguchi, R. (1983). The biosynthesis of mycotoxin patulin.Hakkokogaku, 61, 129-137.

Sekiguchi, J., Shimato, T., Yamada, Y., and Gaucher, G.M. (1983).Patulin biosynthesis: Enzymatic and nonenzymatic transformations ofthe mycotoxin (E)-Ascladiol.Appl. Environ. Microbiol. 45, 1939-1942.

Shen, Y. C., Hein, J., Solomon, N. A., Wolfe, S., and Demain, A. L.(1984). Represion of -lactam production in Cephalosporiumacremonium by nitrogen sources. J. Antibiot. 37, 503-512.

Shen, B., and Hutchinson, C. R. (1993). Enzymatic synthesis of a

bacterial polyketide from acetyl and malonyl coenzyme A. Science262, 1535-1540.

Szab, G., Bekeshi, I., and Vitalis, S. (1967). Mode of action of factorC, a substance of regulatory function in cytodifferentiation. Biochem.Biophys. Acta145, 159-165.

Tak, Y., Inouye, Y., Nakamura, S., Allaudeen, H. S., and Kubo, A.(1989). Comparative studies of the inhibitory properties of antibioticson human immunodeficiency virus reverse transcriptase and cellularDNA polymerases.J. Antibiot. 42, 107-115.

Tanaka, H., Kominato, K., Yamamoto, R., Yoshika, T., Nishida, H.,Tone, H., and Okamoto, R. (1994). Synthesis od doxorubicin-cyclodextrin conjugates.J. Antibiot. 47, 1025-1029.

Thompson, J., Cundliffe E., and Stark, M. J. R. (1982). The mode ofaction of berninamycin and mechanism of resistance in producingorganism Streptomyces bernesis. J. Gen. Microbiol.128, 875-884.

Tomich, P. K. (1988). Streptomyces cloning: Possible construction ofnovel compounds and regulation of antibiotic biosynthesis genes.Antimicrob. Agents Chemother. 32, 1472-1476.

Tomoda, H., and Omura, S. (1990). New strtegy for discovery of

enzymes inhibitors: Screening with intact mammalian cell or intactmicroorganisms having special functions.J. Antibiot. 43, 1207-1222.

Troast, T., Hitchcock, M. J. M., and Katz, E. (1980). Distinctkinureninase and hydroxykinunerinase enzymes in an actinomycin-producing strain ofStreptomyces paravulus. Biochem. Biophys. Acta612, 97-106.

Umezawa, K., Aoyagi, T., Suda, D., Hamada, M., and Takeuchi, T.(1976). Bestatin, an inhibitor of aminopeptidase B, produced byactinomycetes.J. Antibiot. 30, 170-173.

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33/67

Vining, L. C. (1979). Antibiotic tolerance in producer organisms.Advance Appl. Microbiol. 25, 147-168.

Vining, L. C., and Westlake, D. W. S. (1964). Chloramphenicol. In:"Biotechnology of Industrial Antibiotics". (E. J. Vandamme, Ed.). pp.387-411. Marcel Dekker, Inc, New York.

Vo ek, J., urdov, E., Jechov, V., Lenc, B., and Ho lek, Z. (1983). Electron-cytochemical demonstration of polyphosphates and theappropriete phosphates in the glycocalyx ofStreptomycesaureofacien. Cuerrent Microbiol.gy 8, 31-36.

Walker, M. S., and Walker, J. B. (1971). Streptomycin biosynthesis.J.Biol. Chem., 246, 7034-7040.

Walker, J. B. (1975). ATP: Streptomycin 6-phosphotransferase. In:

"Methods in Enzymology"(J. H. Hash, Ed.), 43, 428-470.Weiser, J., Mikulk, K., and Bosh, L., (1981). Studies on the elongationfactor Tu from Streptomyces aureofaciens. Biochem. Biophys. Res.Commun. 99, 16-20.

Westley, J. (1977). Polyether antibiotics: Versatile carboxylic acidionophores by Streptomyces. Adv. Appl. Microbiol. 22, 177-223.

Yarbrough, G. G, Taylor, D. P., Rowlands, R. T., Crawford, M. S. andLasure, L. L. (1993). Screening microbial metabolites for new drugs-theoretical and practical issues.J. Antibiot.46, 535-544.

Zmijewski Jr., M. J., and Fayerman, J. T. ( 1995). Glycopeptides. In:"Genetics and Biochemistry of Antibiotics Production" (L. C. Viningand C. Stuttard, Eds.), pp. 269-281, Butterworth-Heinemann, Boston.

Figures.

Fig. 1. Gramicidin AFig. 2. Gramicidin SFig. 3. BacitracinFig. 4. Bacitracin synthetaseFig. 5. Penicillins

Fig. 6. CephalosporinsFig. 7. Clavulanic acidFig. 8. VancomycinFig. 9. 6-methyl salicylic acid synthetaseFig. 10. TetracyclinesFig. 11. Tetracycline biosynthesisFig. 12. Tetracycline biosynthesisFig. 13. AnthracyclinesFig. 14. ErythromycinsFig. 15. Tylosin and RelomycinFig. 16. Nystatins

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Fig. 17. AvermectinsA - R5= OCH3; B - R5= OH; 1 - X= -CH=CH-; 2 = X= -CH2-

CHOH-;

a - R26= C2H5; b - R26= CH3Fig. 18. ChloramphenicolFig.19. StreptomycinesFig. 20. 6-aminopenicillanic acid and 7-aminocephalosporanicacidFig. 21. Parameters of an industrial fermentation ofS.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 BFig. 22. Factor AFig. 23. Factor B

Adv. Appl. Microbiol. 47, 113-156, 2000.

Bioactive Products from

Streptomyces

VLADISLAV B HALInstitute of Microbiology

Academy of Sciences of the Czech RepublicPrague, Czech Republic

I. IntroductionII. Chemistry and biosynthesis

A. Peptide and peptide-derivative antibioticsB. Polyketide derivatives

C. Other groups of bioactive productsIII. Genetics and molecular genetics

B. Preparation of high production microorganismsB. Genetic manipulation of secondary metabolites producers

IV. Obtaining new bioactive secondary metabolitesA. Isolation from natural resourcesD B. Producers of bioactive compoundsE C. ScreeningD. Semisynthetic and synthetic bioactive productsE. Hybrid bioactive products and combinatorion biosynthesis

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V. Regulation of secondary metabolites productionA.Growth phases of microbial cultureB. Control of fermentation by basal nutrients

C. How signals from the medium are receivedD. Regulation by low molecular compoundsE. AutoregulatorsF. Regulation by metal ions

VI. Resistance to secondary metabolitesA. Resistance of bioactive secondary metabolites producersB. Resistance in pathogenic microorganisms

VII. References

I. Introduction

C. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS

Medicine of twentieth century, especially its second half, wastransformed by the discovery of antibiotics and other bioactivesecondary metabolites produced by microorganisms. Antibiotics aredefined as microbial products that inhibit the growth of othermicroorganisms. After the antibacterial effect of penicillin had beenobserved by Fleming, a number of other antibiotics were discovered,mainly those produced by soil Streptomyces and moulds. Moreover, abroad spectrum of natural products having other effects on living

organisms were found in microorganisms. In addition to standardantibiotics, the following compounds have also been found:coccidiostatics used in poultry farming, antiparasitic compounds witha 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 andpesticide activities and are biodegradable. However, microbialherbicides and pesticides only exceptionally used (e.g. bialaphos) dueto 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 ascertain enzyme activities in human metabolism that cause illness.Many enzyme inhibitors are protease inhibitors, variously activeagainst pepsin, papain, trypsin, chymotrypsin, catepsin, elastase,renin, etc. Inhibitors of glucosidases, cyclic AMP phosphodiesterase,different carbohydrases, esterases, kinases, phosphatases, etc. havebeen also isolated from Streptomyces. The enzyme inhibitors thatblock synthesis of cholesterol are also important. Other exhibit theimmunosuppressive effects, the most famous of them beingcyclosporin A (a cyclic undecapeptide) produced by filamentous

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fungi. Some macrolide antibiotics, isolated from Streptomyces, arealso immunosuppressives.

Several thousands biologically active compounds have been

deseribed and each year new compounds are isolated frommicroorganisms. Microorganisms are a virtually unlimited source ofnovel chemical structures with many potential therapeuticapplications.

The therm "secondary metabolite" used for some microbialproducts BuLock (1961) and suitability of this therm discusedBennett and Bentley (1989). Secondary metabolites are meantcompounds that the microorganism can synthesize but they are notessential for basic metabolic processes such as growth andreproduction. Nevertheless many secondary compounds function asthe so-called signal molecules, used to control the producers

metabolism. Another function attributed to antibiotics is asuppression of competing microorganisms in the environmentwhereby the antibiotic-producing microorganisms have an advantagein competing for nutrients with the other microorganisms.

The production of secondary metabolites in microorganismsisolated from nature is rather low in most cases.To be usable for thecommercial production of secondary

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Bioactive Products From Streptomyces