In the last century, microorganisms exceeded plants in their importance as sources of biologically active substances and they still remain the source of an inexhaustible number of compounds with a variety of biological functions (Běhal 2001). The most important of these biologically active compounds are antibiotics that suppress the growth of pathogenic microorganisms such as bacteria, fungi and viruses. Although several thousands of antibiotics have been discovered and put to use, new ones are being searched for because the pathogenic microorganisms acquire resistance to existing substances of this class. The most common source of antibiotics is soil microorganisms, in particular streptomycetes, molds and fungi. The phenomenon of resistance has boosted the efforts to find antibiotics with new mechanisms of action. Antibiotics isolated from natural sources are being variously modified to produce compounds that would overcome the resistance of strains insensitive to natural derivatives (Běhal 2000). Methods of molecular genetics allow the acquisition of new biologically active substances that do not occur in the nature by making use of a combination of structural genes from different microorganisms. This review sums up the latest results achieved in this field.

ANTIBIOTIC SYNTHESIS

Antibiotics belong to secondary metabolites; these substances are produced by microorganisms but are not essential for their basic metabolic processes such as growth and reproduction. If they are produced in quantities exceeding several times their amounts produced under natural conditions, we talk about overproduction. Secondary metabolites are synthesized from building blocks identical with those used for the synthesis of other parts of the microbial cell, viz. amino acids, thioester acyls and saccharides. Genes encoding the enzymes participating in the biosynthesis of secondary metabolites are mostly located on chromosomes and are called structural genes (Diez 1997). Genes for resistance against the organism’s own antibiotics are located in the immediate vicinity of structural genes. The level of antibiotic production depends not only on the number of copies of structural genes in the cell but predominantly on mechanisms regulating the expression of structural genes (Běhal 1986). Analysis of DNA of genes responsible for penicillin production in Penicillium chrysogenusm showed a several-fold amplification of the genes of isopenicillin-A-synthetase (Smith et al. 1989) but the production of the antibiotic was not proportional to the multiple of structural genes. Production of secondary metabolites involves an important role of regulatory proteins that specifically bind to promoters and trigger thereby the expression of genes of secondary metabolism (Cu et al. 1995; Feng et al. 1995). The production of secondary metabolites is a complex problem; many factors limit antibiotic overproduction and, if one of them is eliminated, another takes up its role.

Isolation of enzymes of secondary metabolism and identification of the appropriate structural genes on the chromosome has allowed their transfer into other microorganisms.

POLYKETIDE BIOSYNTHESIS

Polyketides represent a large group of antibiotics synthesized by condensation of thioester acyls, especially acetyl-CoA, malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA. The enzyme complex that catalyzes the recurrent decarboxylative condensation of acyl thioesters is called polyketide synthase (PKS). The mode of condensation of the polyketide chain is analogous to the biosynthesis of fatty acids by fatty acid synthetase but differs from it in that the extension of the nascent chain by one acyl is not accompanied by the total reduction of the keto group. This ensures a large variability of structures of the resulting products. Proteins representing PKS in microorganisms producing polyketides constitute 1 – 5 % of total cell proteins (Khosla et al. 1999). In the case of polyketide overproduction the proportion of PKS in total proteins will probably be still higher. The PKSs found in individual polyketide producers can be divided into three highly similar groups. The three types of PKSs are PKSs I, which participate in the biosynthesis of macrolide antibiotics, and PKSs II which produce aromatic polyketides and PKSs III, similar to PKSs II, used as terminal group aromatic substances.

PKS I

This PKSs type are multifunctional enzymes composed of separate groups of active centers. After each acyl condensation, these centers are the site of reactions leading to the production of the corresponding polyketide (McDaniel et al. 1994; Bentley and Bennett 1999). The best studied is the enzyme complex of deoxyerythronolide B- synthase (DEBS) of Saccharopolyspora erythreus, which synthesizes the aglycone of erythromycin (Fig. 6). The size of the DNA cluster encoding the synthesis of 6-dEB (eryA) is 32-35 kb. Sequencing of the 35 kb segment showed that DEBS is composed of three multifunctional proteins (DEBS1, DEBS2 and DEBS3), each of which contains two modules. In addition, it includes a loading module containing acyl transferase (AT), an acyl carrier protein (ACP) located before the first module, and an off-loading domain containing thioesterase/cyclase (TE) (Fig. 1) (Pieper et al. 1995; Donadio et al. 1991; Marsden et al. 1998). The terminal acyl condenses with an acyl bound to ACP of another subunit and the ensuing b-ketoacyl-ACP is transformed on the same subunit in a manner corresponding to the final product. The transformed acyl is transferred to the next subunit, where elongation by another acyl takes place and the terminal acyl is again transformed in a way specific for the given subunit. The elongation process then takes place on other modules until the resulting aglycone reaches its final length (McDaniel et al. 1997). Enzymes participating in the transformation of b-ketoacyls are ACP, acyl transferase (AT), dehydratase (DH), enoyl reductase (ER), ketoreductase (KR), ketoacyl-ACP-synthase (KS) and thioesterase/cyclase (TE). Whereas other enzymes are present in all modules, TE is located as the last subunit only on the sixth module and obviously tranfers the resulting acyl-CoA to CoA, where cyclization to 6-dEB takes place under the catalysis of a cyclase located 4 kb downstream of module 6 (Weber et al. 1991). DEBS was isolated together with TE from S. erythrea and its kinetic properties were determined by Bycroft et al. ( 2000). Pieper et al. (1995) carried out an in vitro synthesis of 6-dEB. A homology was found between the PKS-encoding cluster from the myxobacterium Sorangium cellulosum So ce26, a producer of the antifungal macrolide antibiotic soraphen A and the cancerostatics epothilons (Fig. 2) and PKS-encoding genes from Streptomyces violaceoruber. In particular, similarity was found between domains corresponding to AT, ACP, KR and DH. The epothilon-synthesizing PKS from S. cellulosum is composed of nine modules, a loading module and a terminal enzyme, P450 epoxidase, which catalyzes the epoxidation of deepoxyepothilons to epothilons (Tang et al. 2000). Hence in myxobacteria macrolides are synthesized by PKS of type I (Schupp et al. 1995). The polyketide synthase of the avermectin (Fig. 3) producer S. avermitilis is composed of 12 modules. Two genes for postpolyketide modification are located between two sets of genes for polyketide synthase; one of them codes for cytochrome P450 hydroxylase, which probably catalyzes the formation of a furan ring between C6 and C8a (Ikeda et al. 1999). The flexibility of such systems renders possible a combination of structural genes of different PKSs and thus also the formation of hybrid multienzymes which can synthesize new antibiotics (Marsden et al. 1998).

PKS II

Aromatic polyketides (tetracyclines, anthracyclines) are synthesized by a mechanism slightly different from that used for the above macrolide compounds. The polyketide synthases synthesizing aromatic polyketides (aromatic PKSs) and found mainly in actinomycetes are multienzymes that are the site of all condensation, reduction and cyclization reactions. The structures of aromatic polyketides are multitudinous and their synthesis thus involves different polyketide synthases. However, every aromatic PKS contains a minimum set of three proteins (minimum PKS) necessary for in vivo synthesis of polyketides. They are a bifunctional ketide synthase/acyl transferase (KS/AT), a chain length-determining protein (CLF) and ACP. Most aromatic PKSs contain also special ketide reductase (KR) and cyclase (CYC) enzymes (McDaniel et al. 1994). An important enzyme is KR that catalyzes the regio-specific reduction of C-9 carbonyl of the polyketide chain (Fig. 4) irrespective of the chain length (Khosla et al. 1999). In aromatic PKSs, KRs obviously act also on fully synthesized polyketide chains. In addition to the minimum PKS and KR, bacterial aromatic PKSs contain also homologous proteins called aromatases and cyclases (ARO/CYC). The mechanism of their action is not yet known but they obviously play an important role in the formation of the first aromatic ring of aromatic polyketides. A combination of genes coding for subunits of different aromatic PKSs permits also the synthesis of new hybrid antibiotics.

PKS III

The type III PKSs were studied predominantly in plants (Jez et al. 2002) and they are not well investigated. They are homodimeric enzymes that orchestrate a series of acyltransferase, decarboxylation, condensation, cyclization, and aromatization reactions at two functionally independent active sites. They accepts disparate CoA starter molecules, including aromatic CoA thioesters.

GENETIC ENGINEERING

Methods of genetic engineering are used to combine PKSs from two or more different antibiotic producers. Parts or the whole gene for actinorhodine biosynthesis from Streptomyces coelicolor A3 were transferred into the medemycin producer Streptomyces sp. AM7161 to carry out the synthesis of new metabolites mederhodine A and B (Fig. 5). In contrast to medemycin, these derivatives were hydroxylated at the C-6 position of the naphthoquinone skeleton by hydroxylase from a DNA fragment arriving from S. coelicolor (Malpartida and Hopwood 1984). The production of the mederhodines was relatively high and stable. When the whole actinorhodine gene was introduced into Streptomyces sp. AM 7161, mederhodines were not synthesized, the only products being actinorhodine and medemycin. The antimicrobial activity of mederhodine A was the same as that of medemycin.

The first two enzymes of the DEBS loading module, AT and ACP, were replaced by the same enzymes from the PKS loading module of the avermectin producer S. avermitilis. The expression of this hybrid PKS in the erythromycin producer S. erythreus made it possible to synthesize alternative erythromycins since the S. avermitilis PKS accepts more than 40 alternative carboxylic acids as a starting units (Marsten et al. 1998). While the S. erythreus with the unhybridized PKS produced only three types of erythromycins, A, B and D, S. erythreus with the hybridized PKS produced erythromycins in whose molecule the ethyl group in position R₁ was substituted by isopropyl or sec-butyl group (Fig. 6). If the culture medium for this recombinant stran S.erythreus was during cultivation supplemented by short-chain fatty acids (cyclopentane carboxylic acid, 1- cyclopentene carboxylic acid, cyclopentene carboxylic acid, cyclobutane carboxylic acid, 3-methylcyclobutane carboxylic acid, 3-furoic acid, thiopene 3-N-acetyl cysteamine ester, methylthiolactic acid), the cultivation yielded new biologically active erythromycins substituted with the added acyls at C-13 (Pacey et al. 1998). When these short-chain fatty acids were added to the wild-type strain, they were not incorporated into the erythromycin molecule. The erythromycin analogues so obtained exhibited different biological activities against Pasteurella multocida, Escherichia coli and Staphylococcus aureus; these activities were mostly lower than those of unmodified erythromycins, with the exception of an analogue with cyclopentane carboxylic acid as substituent, which showed a marked activity against P. multocida and E. coli.

In an analogous approach, the b-ketide reductase domain in module 2 of erythromycin PKS was substituted by b-ketide reductase and dehydratase domain from rapamycin PKS. The resulting chimeric multifunctional enzyme catalyzed a b-ketoreduction and regio-specific dehydratation of the covalent bond in the triketide derivatives; the product arising by these reactions was transferred to another module and modified by erythromycin PKS in a normal manner (Khosla et al. 1999).

When the first three DEBS modules were expressed in Streptomyces coelicolor CH999, the products were the tetraketide lactone CK13a and a decarboxylated tetraketide hemiketal CK13b (Fig. 7). If, prior to the expression of the three modules in S. coelicolor, the DH/KR from the second module of DEBS PKS was substituted by the DH/KR from the fourth module of rapamycin PKS, the synthesized products included dehydrated tetraketides KOS009-7a and KOS0097b (Fig. 8) (McDaniel et al. 1997).

The gene for the acyl-modification of macrolide antibiotics, 4´´-O-acyltransferace gene from Streptomyces thermotolerans, was introduced into the tylosin producer Streptomyces fradiae together with a regulatory gene. The transformant produced 3-O-acetyltylosin and 3-O-acetyl-4´´-acetyltylosin (Fig. 9). In addition, the medium was found to contain a small amount of 3-O-acetyl-4´´-isovaleryltylosin. When the medium was supplemented with L-leucine, a precursor of isovaleryl-CoA, the latter substance became the main product (Arisava et al. 1996).

The gene encoding carminomycin 4-O-methyltransferase in Streptomyces peucetius was introduced into the epelmycin producer Streptomyces violaceus. The transformant produced, along with epelmycin, a hybrid anthracycline 4-O-methylepelmycin D (Fig. 10) (Miyamoto et al. 2000). As compared with epelmycin, 4-O-epelmycin inhibited somewhat less the growth of leukemic L1210 cells but was much more active in inhibiting RNA and DNA synthesis in these cells.

A chromosomal fragment containing rubromycin PKS from Streptomyces collinus was transferred into S. coelicolor CH999 and the recombinant strain then produced 5 – 10 new metabolites. The most important of these, collinone (Fig. 11), exhibited antibacterial activity against vancomycin-resistant enterococci but not the antifungal or antiviral activity of rubromycin (Martin et al. 2001).

Aklavinone (Fig. 12) produced by Streptomyces galilaeus is the precursor of aglycones of a number of anthracyclines. S. galilaeus is thus a suitable acceptor of DNA segments from producers of other antibiotics and is capable of producing hybrid antibiotics. Hybridization of structural genes of S. galilaeus and Streptomyces purpurascens producing ε-rhodomycinone and β-rhodomycinone (Fig. 13) gave new glycosides 10-demethoxycarbonylaklavinone and 11-deoxy-β-rhodomycinone (Fig. 14); the original S. galilaeus products were thus modified in positions 10 and 11 by genes from S. purpurascens. The hybrid strain produced also the original substances aklavinone, ε-rhodomycinone and β-rhodomycinone. 11-Deoxy-β-rhodomycinone exhibited cytotoxic activities against mouse L1210 leukemic cells (Niemi et al. 1994).

The gene for 11-hydroxylase from the doxorubicin producer Streptomyces peuceticus ATCC 27952 was introduced into the aklavinone producer Streptomyces galilaeus ATCC 31133. The transformed strain produced a number of red colored metabolites such as 11-hydroxyaclacinomycin A, B, T and X (Fig. 15). 11-Hydroxyaclacinomycin X displayed a strong cytostatic activity against human cancer cell lines, especially leukemic and melanoma ones (Kim et al. 1996). Since anthracyclines have a complex chemical structure and are efficient cancerostatic, the preparation of new derivatives by hybridization is a promising way for acquiring new substances with cancerostatic effects.

Apart from hybrid polyketides, also hybrid peptides with activities different from those of original peptides have been prepared. A hybrid of the antimicrobial peptide cecropine A and the peptide melittin contained in bee venom exhibited high activity against both Gram-negative and Gram-positive bacteria, especially against Staphylococcus aureus, and was also effective against malaria. Unlike melittin, the hybrid did not show a hemolytic action (Boman et al. 1989). A combination of seven amino acids derived from a protein produced by larvae of the silk moth Hyalophora cecropia and eight amino acids from melittin afforded a peptide with a higher activity against anaerobic bacteria, which was also free of the undesirable hemolytic properties of melittin (Oh et al. 2000). Hybrid peptides were also found to enhance induction of cell immunity (Moroi et al. 2000). Other analogous peptides have been described in patent literature.