Download Hybrid Antibiotics

Folia Microbiol. 48, 17-25 (2003)
Hybrid Antibiotics
V. Běhal
Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague,
Czechia
e-mail [email protected]
Abstract. Hybrid antibiotics that do not occur in nature have been obtained by combining
structural genes of antibiotic producers. Some of these substances were effective against
pathogenic microorganisms resistant against antibiotics produced by the parent strains. The
majority of hybrid antibiotics were obtained by combining genes encoding polyketide
synthases. Hybrid peptides with new biological properties have also been synthesized.
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INTRODUCTION
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.
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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
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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.
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POLYKETIDE BIOSYNTHESIS
Polyketides represent a large group of antibiotics synthesized by condensation of
thioester acyls, especially acetyl-CoA, malonyl-CoA, methylmalonyl-CoA and ethylmalonylCoA. 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 -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 -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 acylCoA 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 PKS2
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 lengthdetermining 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.
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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
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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 -ketide reductase domain in module 2 of erythromycin
PKS was substituted by -ketide reductase and dehydratase domain from rapamycin PKS.
The resulting chimeric multifunctional enzyme catalyzed a -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 3O-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 Lleucine, 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.
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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-βrhodomycinone 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 11hydroxyaclacinomycin 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 Gramnegative 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.
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CONCLUSIONS
As shown on the examples mentioned above, hybridization of structural genes from
different producers of biologically active substances may furnish a number of new products.
Their number will undoubtedly increase with the extending knowledge in the field and
increasing number of laboratories equipped with suitable methodology for their preparation.
Biologically active secondary metabolites produced by microorganisms usually have complex
structures so that their chemical synthesis is difficult and costly. The chance of derivatives of
biologically active compounds displaying desirable biological activities can be expected to be
higher than with new isolates from nature.
The sequencing of the human genome will facilitate more efficient treatment of
diseases. A number of diseases have genetic basis but are triggered by the action of external
factors. For instance, a faulty function of T-cells that liquidate all substances recognized as
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foreign, including viruses and bacteria, can give rise to autoimmune diseases such as
rheumatoid arthritis, gastritis, multiple sclerosis, and primary biliary cirrhosis. Which disease
will appear depends on the agent attacking the T-cells. Instead of searching for substances
curing separate diseases, it is more advantageous to look for substances eliminating their
causes. Production of hybrid microbial metabolites is one of the possibilities of obtaining
substances with desirable structures. The preparation of hybrid substances with biological
activity can thus be expected to have a wide application.
Acknowledgement
This work was supported by the Grant Agency of the Czech Republic (grant no.
204/01/1004)
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Obrázky
Fig.1. Model of modular polyketosynthase synthesizing 6-deoxyerythronolide B.
DEBS1, DEBS2 and DEBS - multifunctional enzymes, AT- acyltransferase, KS - β-ketoacylACPsynthase, ACP - acyl carrier protein, KR - β-ketoreductase, DH - dehydratase, ER enoylreductase, TE - thioesterase.
Fig. 2. Epothilons
Fig. 3. Avermectin B1a
Fig. 4. Regiospecific reduction of th C9 carbonyl of the poly-β-ketone backbone by
ketoreductase.
Fig. 5. Production of a hybrid antibiotic mederhodin A
Fig. 6. Production of new erythromycins by hybrid 6-deoxyerythronolide B synthase.
Acyltransferase and acyl carrier protein of loading modul origin from S. avermitilis.
1 - (eryA) - R1=Et, R2=OH, R3=CH3
2 - (eryB) - R1=Et, R2=H, R3=CH3
3 - (eryD) - R1=Et, R2=H, R3=H
Fig. 7. Products of expression of the first three DEBS modules in S. coelicolor CH999.
Fig. 8. Products of expression of the first three DEBS modules where DH/KR of module 2
were substituted by DH/KR from module 4 of rapamycin PKS in S. coelicolor.
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Fig. 9. Acylderivatives of tylosin
Fig. 10. 4-O-epelmycin D
Fig. 11. Collinone
Fig. 12. Aklavinone
Fig. 13. ε-Rhodomycinone and β-Rhodomycinone
Fig. 14. 10-demethoxycarbonylaklavinone and 11-deoxy-β-rhodomycinone
Fig. 15. 11-hydroxyaclacinomycines
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