Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8, D-72076 Tübingen, Germany1
Author for correspondence: Shu-Ming Li. Tel: +49 7071 2976995. Fax: +49 7071 295250. e-mail: shuming.li{at}uni-tuebingen.de
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ABSTRACT |
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Keywords: coumermycin A1, novobiocin, aldolase, retro-aldol reaction
The GenBank accession number for the sequence of cosmid K1F2 is AF329398.
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INTRODUCTION |
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The affinity of the aminocoumarin antibiotics for bacterial gyrase is extremely high. The inhibition constant (Ki) values of these antibiotics are in the 10 nM range, i.e. two orders of magnitude lower than those of modern fluoroquinolones. This makes the aminocoumarins very interesting starting materials for the development of new antibacterial compounds.
Previously, our group has identified the biosynthetic gene clusters for novobiocin from Streptomyces spheroides NCIMB 11891 (Steffensky et al., 2000a ) and for coumermycin A1 from Streptomyces rishiriensis DSM 40489 (Wang et al., 2000
). In the present study, we report the identification of the clorobiocin biosynthetic gene cluster from Streptomyces roseochromogenes DS 12.976. A comparison of the gene clusters for clorobiocin, novobiocin and coumermycin A1 showed that the structural differences between the three antibiotics corresponded well to the differences in the organization of their respective biosynthetic gene clusters. Furthermore, the very stringent organization of the biosynthetic genes encoding the three different aminocoumarin antibiotics into modules, each of which carries the complete genetic information required for the biosynthesis of the respective antibiotic, offers excellent prospects for the production of novel aminocoumarins using a combinatorial biosynthetic approach.
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METHODS |
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Escherichia coli XL-1 Blue MRF' (Stratagene) was grown in liquid or on solid LuriaBertani medium at 37 °C (Sambrook & Russell, 2001 ). SuperCos-1 was purchased from Stratagene. pBSKT, an integrative vector carrying carbenicillin and thiostrepton resistances, was described by Lombo et al. (1997)
.
Carbenicillin (50 µg ml-1) and thiostrepton (50 µg ml-1) were used in the medium for selection of recombinant plasmids and strains.
Genetic procedures.
Standard methods for DNA isolation and manipulation were performed as described by Kieser et al. (2000) . DNA fragments were isolated from agarose gels using a NucleoSpin 2 in 1 extraction kit (Macherey-Nagel). Isolation of cosmids and plasmids was carried out with ion-exchange columns (Nucleobond AX kit; Macherey-Nagel). Genomic DNA was isolated from Streptomyces strains by lysozyme treatment and phenol/chloroform extraction.
Construction and screening of the cosmid library.
Chromosomal DNA of S. roseochromogenes was partially digested with Sau3AI, dephosphorylated and then ligated into the BamHI sites of SuperCos-1. The ligation products were packaged with Gigapack III XL (Stratagene, Heidelberg; Germany) and transduced into E. coli XL-1 Blue MRF'.
Southern blot analysis was performed on Hybond-N membranes (Amersham) with digoxigenin-labelled probes by using the DIG high prime DNA labelling and detection kit II (Roche Molecular Biochemicals). Two probes, one containing part of the dTDP-glucose 4,6-dehydratase gene novT (Steffensky et al., 2000a ) and one containing a 1·58 kb SphIBamHI fragment of the novobiocic acid synthetase gene novL (Steffensky et al., 2000b
), were used for hybridizations.
DNA sequencing and computer-assisted sequence analysis.
Double-stranded sequencing of the entire cosmid K1F2 (carrying an insert of 42291 bp) was performed by the dideoxynucleotide chain termination method on a LI-COR automated sequencer (MWG-Biotech) using a shotgun library with DNA fragments of approximately 1·52·0 kb in length.
The DNASIS software package (version 2.1; Hitachi Software Engineering) was used for sequence analysis. Amino acid sequence homology searches were carried out in the GenBank database by using the BLAST program (release 2.0).
Construction of pFP02 for in-frame gene inactivation.
For inactivation of cloR in S. roseochromogenes, the fragments cloR-1 (1282 bp) and cloR-2 (1301 bp) were amplified by PCR. Primer pair cloR-1/HindIII (5'-GTCACCGGAAGCTTTGCCTG-3') and cloR-1/PstI (5'-GCATGTTCTGCAGAGCCTTG-3') was used to amplify cloR-1; primer pair cloR-2/PstI (5'-GCCTGCACTGCAGGCCCCAA-3') and cloR-2/BamHI (5'-TCGTAGGATCCTCCCGTCGTC-3') was used to amplify cloR-2. Restriction sites introduced into the primers are shown in bold in the aforementioned sequences. cloR-1 was digested with HindIII and PstI and cloned into the corresponding sites of vector pBSKT, a pBluescript SK(+) derivative containing carbenicillin and thiostrepton resistances, resulting in pFP01. cloR-2 was digested with PstI and BamHI and ligated into the same sites of pFP01 to give pFP02.
Transformation of S. roseochromogenes and selection of recombinant mutants.
Transformation of S. roseochromogenes with pFP02 was carried out by polyethylene glycol-mediated protoplast transformation (Kieser et al., 2000 ). For the preparation of protoplasts, mycelia of S. roseochromogenes were grown in CRM medium containing 10·3% sucrose, 2·0% tryptic soy broth, 1·0% MgCl2.6H2O, 1·0% yeast extract and 0·75% glycine (pH 7·0) for 48 h. The mycelia were then harvested and incubated in 5 ml P (protoplast) buffer (g mycelium)-1 containing 1 mg lysozyme ml-1 for 3060 min at 30 °C.
For transformation of S. roseochromogenes, pFP02 was mixed with 200 µl P-buffer containing 1x109 S. roseochromogenes protoplasts and 500 µl T (transformation) buffer containing 50% (w/v) polyethylene glycol 1000 (Roth). The resulting suspension was plated onto R2YE agar. After incubation for 20 h at 30 °C, the plates were overlaid with 3 ml of soft R2YE agar containing a total of 500 µg thiostrepton, for selection of the recombinant mutants.
After the transformation of S. roseochromogenes protoplasts with pFP02, thiostrepton-resistant colonies were obtained. The single-cross-over mutant RSCO2 was grown in the absence of thiostrepton, allowed to sporulate and then examined for loss of resistance to thiostrepton due to double-cross-over events. Two mutants, named RDCO30 and RDCO32, were examined further. Chromosomal DNA from wild-type S. roseochromogenes, as well as from mutants RSCO2, RDCO30 and RDCO32, was digested with SacII and hybridized with a probe containing part of cloR. A band of approximately 1·1 kb in size was detected upon hybridization of the wild-type S. roseochromogenes DNA with the probe, whereas hybridization of the chromosomal DNA from mutant strain RDCO30 with the probe produced the expected 2·2 kb band, which corresponded to the in-frame deletion of cloR (Fig. 2).
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Analysis of secondary metabolites.
The bacterial culture (20 ml) was acidified to pH 4 with HCl and extracted twice with an equal volume of ethyl acetate. After centrifugation, the solvent was evaporated and the dried extract was resuspended in 1 ml methanol. Metabolites were analysed by HPLC using a Multosphere RP18-5 column (250x4 mm, 5 µm) with a linear gradient from 60 to 100% methanol in 1% aqueous formic acid and detection at 340 nm. Authentic clorobiocin (Aventis) was used as the standard.
For preparative isolation of the secondary metabolites, the fractions from HPLC analysis were collected and the solvent was evaporated. The product was analysed by MS and 1H-NMR. Negative fast-atom bombardment (FAB) mass spectra were recorded on a TSQ70 spectrometer (Finnigan) using methanol as the solvent; peaks A and B gave identical isotopic peaks, characteristic for substances with one chlorine atom, at m/z 697 and 695 [M-H]- that corresponded to the clorobiocin standard. The 1H-NMR spectrum was measured on an AMX 400 spectrometer (Bruker), and peak A gave signals corresponding to those of the clorobiocin standard: p.p.m. (CD3OD, 400 MHz), 7·90 (d, J=9·2 Hz, H-5), 7·76 (d, J=2·5 Hz, H-2'), 7·72 (dd, J=8·4 Hz, 2·5 Hz, H-6'), 7·33 (d, J=9·2 Hz, H-6), 6·90 (d, J=3·6 Hz, H-3'''), 6·84 (d, J=8·4 Hz, H-5'), 5·94 (d, J=3·6 Hz, H-4'''), 5·73 (d, J=1·8 Hz, H-1'), 5·71 (dd, J=10·3 Hz, J=2·9 Hz, H-3'), 5·35 (broad t, J=7·1 Hz, H-8'), 4·34 (t, J=2·7 Hz, H-2'), 3·72 (d, J=10·3 Hz, H-4'), 3·52 (s, 3H-8'), 3·34 (d, J=7·1 Hz, 2H-7'), 2·29 (s, 3H-6'''), 1·75 (s, 3H-11'), 1·74 (s, 3H-10'), 1·35 (s, 3H-7'), 1·18 (s, 3H-6').
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RESULTS |
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A cosmid library from S. roseochromogenes was constructed in SuperCos-1 and screened with the novT and novL probes. The hybridizing cosmids were analysed by restriction mapping. Four different but overlapping cosmids were obtained which covered a continuous 55 kb region of the chromosome. Cosmid K1F2 was sequenced on both strands. From the sequence of this cosmid, 36 ORFs were identified; 27 of these showed striking similarity to genes of the novobiocin and/or coumermycin A1 biosynthetic gene clusters. In addition, a partial sequence of the aminocoumarin-resistance gene (gyrBR) was found at the 3' end of the clorobiocin gene cluster. Strikingly, in the gene clusters of coumermycin A1, novobiocin and clorobiocin, the corresponding ORFs were arranged in exactly the same order and oriented in the same direction (Fig. 1).
Table 1 lists the homologies found between the genes in cosmid K1F2 and the genes of the novobiocin and coumermycin A1 clusters, as well as the homologies of the K1F2 genes to other sequences found in GenBank. The sequence of cosmid K1F2 has been deposited in GenBank under accession no. AF329398.
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Genes presumably involved in the formation of the pyrrole ring
Clorobiocin and coumermycin A1 contain pyrrole carboxylic acid rings attached to position 3 of their deoxysugar moieties (Fig. 1). Novobiocin contains a carbamyl group at the corresponding position.
These structural similarities and differences between the three antibiotics are reflected in the organization of their respective gene clusters (Fig. 1). Downstream of the glycosyltransferase gene novM, the novobiocin cluster contains a gene (novN) with homology to those encoding carbamoyl transferases, whereas in the same relative position of the clorobiocin and coumermycin clusters, a group of seven genes is found (cloN1N7 or couN1N7, respectively) which show very high homology between the two clusters (on average 87% identity). These genes can be assigned to pyrrole biosynthesis, which has been elucidated on the genetic and biochemical level: cloN3, cloN4 and cloN5 show sequence similarities to pltE, pltF and pltL, respectively, which are involved in the biosynthesis of the pyrrole moiety of pyoluteorin in Pseudomonas fluorescens Pf-5 (Nowak-Thompson et al., 1999
) and to redW, redM and redO, respectively, which are involved in the biosynthesis of the pyrrole moiety of undecylprodiginin in Streptomyces coelicolor (Cerdeno et al., 2001
). PltF and RedM convert L-proline into its acyl adenylate (Fig. 3
) and the small proteins PltL and RedO act as peptidyl carrier proteins (PCPs) (Thomas et al., 2002
). Therefore, the same functions may be assigned to the homologous proteins CloN4 and CloN5, respectively. CloN3, like CouN3, PltE and RedW, shows homology to flavine-dependent acyl-coenzyme A dehydrogenases. PltE and RedW catalyse the dehydrogenation of the PCP-bound proline (Thomas et al., 2002
). The resulting pyrroline derivative (presumably
2) undergoes spontaneous oxidation to the aromatic pyrrole derivative (Fig. 3
).
CloN6 (CouN6) belongs to the BchE-like/methyltransferase subgroup of radical SAM proteins, which has recently been identified using bioinformatic techniques (Sofia et al., 2001 ). We suggest that CloN6 catalyses the transfer of a methyl group to position 5 of the pyrrole-2-carboxylic acid. cloN2 (couN2) shares homology with dpsC, which encodes an enzyme with acyltransferase activity. cloN7 and/or cloN2 may be involved in the transfer of the activated pyrrole-2-carboxylic acid to the 3-OH of the deoxysugar moiety. The small ORF cloN1 (encoding 95 aa) does not show homology to other database entries, and its function remains unknown at present.
Genes presumably involved in the biosynthesis of the aminocoumarin ring (Ring B)
The genes for the biosynthesis of the characteristic aminocoumarin ring must be present in all three gene clusters; hence, a comparison of the three gene clusters presents an obvious method for identifying possible candidate genes involved in the biosynthesis of this ring. In the clorobiocin cluster, cloHIJK showed, on average, 85% homology to the corresponding genes in the novobiocin (novHIJK) and coumermycin A1 (couHIJK) clusters (Table 1). It appears likely that the products of these genes are involved in the formation of the aminocoumarin ring from tyrosine (see Discussion).
Clorobiocin contains a chlorine atom at position 8 of the aminocoumarin ring, whereas novobiocin and coumermycin A1 contain a methyl group at the same position (Fig. 1). This structural difference of the antibiotics is perfectly reflected in the organization of their gene clusters: the novobiocin and coumermycin clusters contain a C-methyltransferase gene, novO or couO, respectively, whereas in the clorobiocin cluster, clohal, a homologue of non-haem halogenase genes, is found at the same relative position.
Genes presumably involved in the biosynthesis of the 3-dimethylallyl-4-hydroxybenzoic acid (Ring A)
Clorobiocin and novobiocin contain a prenylated 4-hydroxybenzoate moiety (Ring A). Coumermycin A1 contains a pyrrole dicarboxylic acid moiety instead, linking the two aminocoumarin rings of this molecule (Fig. 1). The aromatic nucleus of Ring A of clorobiocin and novobiocin is derived from tyrosine (Bunton et al., 1963
; Kominek & Sebek, 1974
), but the exact reaction sequence is unknown.
Sequencing of the clorobiocin gene cluster revealed two genes that are also present in the novobiocin cluster, but which are not present in the coumermycin cluster, i.e. cloQ and cloR. This fact drew us to the hypothesis that these genes may be involved in Ring A biosynthesis.
cloQ and cloR, like novQ and novR, show transcriptional coupling (i.e. the stop codon of cloQ is fused with the start codon of cloR) and are likely to be transcribed as a single operon. Unusually large intergenic regions are found upstream and downstream of cloQR (1001 and 830 bp, respectively). CloR has 47% identity to a putative aldolase from S. coelicolor. cloQ did not show homologies to other genes in the database, with the exception of novQ.
Generation of a cloR-defective mutant
To test whether cloR was indeed involved in Ring A biosynthesis, a gene inactivation experiment was carried out. An inactivation vector carrying a thiostrepton-resistance gene (pFP02) was constructed, in which the structural gene cloR was disrupted by an in-frame deletion (Fig. 2). The deletion mutant, RDCO30, was subsequently cultured and the ethyl acetate extract of the culture was examined by HPLC for secondary metabolites. As shown in Fig. 4
, the production of clorobiocin was abolished in this mutant. Another thiostrepton-sensitive strain obtained in the screening for double-cross-over mutants, RDCO32, represented a reversion to the wild-type (Fig. 2
), and showed clorobiocin production identical to that of the wild-type strain (data not shown).
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Besides the major peak for clorobiocin (peak A in Fig. 4), a minor peak of identical mass (peak B) was detected both in the wild-type and the complemented mutant strain. This substance is likely to represent an isomer of clorobiocin, possibly carrying the pyrrole carboxylic acid moiety in position 2 instead of position 3 of the deoxysugar. Such isomers have been reported previously for novobiocin (Hinman et al., 1957
).
Genes involved in the linkage of Rings A, B and C of clorobiocin
Attachment of the deoxysugar to the 7-OH group of the aminocoumarin ring requires very similar glycosyl transferases in clorobiocin, novobiocin and coumermycin A1 biosynthesis; indeed, three very similar putative glycosyltransferase genes, cloM, novM and couM, are found at the same relative position in all three gene clusters.
In clorobiocin and novobiocin, the aminocoumarin moiety (Ring B) and the prenylated 4-hydroxybenzoate moiety (Ring A) are linked by an amide bond (Fig. 1). It has been demonstrated that the enzyme NovL catalyses this reaction, i.e. the adenylation of the substituted benzoyl moiety and its transfer to the amino group (Steffensky et al., 2000b
). cloL shows high homology to novL and is most probably involved in the formation of the amide bond of clorobiocin.
Resistance and regulatory genes
Downstream of the deoxysugar biosynthesis genes cloSTUVW in the clorobiocin cluster, and similarly at the corresponding position of the novobiocin and coumermycin clusters, a gene encoding an aminocoumarin-resistant gyrase B subunit (gyrBR) is located. This gene has previously been identified as the principal novobiocin-resistance gene in the novobiocin producer S. spheroides (Thiara & Cundliffe, 1988 ).
cloG, novG and couG are homologous to strR, a regulatory gene from the streptomycin cluster. Streptomycin biosynthesis is known to be regulated by -butyrolactones (Horinouchi & Beppu, 1995
). It may, therefore, be speculated that
-butyrolactones are involved in the regulation of the biosynthesis of clorobiocin and other aminocoumarin antibiotics.
cloE has homology to the lmbU gene of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis 78-11. It was suggested that LmbU may have a regulatory function, but no experimental evidence is available so far to support this (Peschke et al., 1995 ).
Genes with unknown function
At present, no function can be suggested for the small ORFs cloY and cloN1, which have homologues in the coumermycin A1 cluster, and for cloZ, which has no homologues in other clusters.
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DISCUSSION |
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The primary metabolic genes found at the 5' end of the clorobiocin cluster (Table 1) suggest that cloE represents the 5' border of the cluster. The gene adjacent to cloE, i.e. ORF9, encodes a putative transposase, and it may be speculated that this gene is related to the introduction of the cluster into the S. roseochromogenes genome. Also at the 3' end, downstream of gyrBR, primary metabolic genes were found, suggesting that the sequence depicted in Fig. 1
comprises all biosynthetic genes of the clorobiocin cluster. However, it cannot be excluded that additional biosynthetic enzymes for clorobiocin formation are encoded at different loci of the genome.
The clorobiocin cluster contains the genes cloHIJK, for which homologues exist in the novobiocin cluster (novHIJK) and the coumermycin cluster (couHIJK). NovH has recently been shown to activate tyrosine by covalent binding to the 4-phosphopantetheinyl cofactor, and the P450 enzyme NovI catalyses the ß-hydroxylation of the activated tyrosine (Chen & Walsh, 2001 ). A central, unresolved question in aminocoumarin biosynthesis is how activated ß-hydroxy tyrosine is then converted to the coumarin ring, especially how the ring oxygen is introduced.
cloJ and cloK, the genes immediately downstream of cloH and cloI, are homologous to novJK and couJK of the novobiocin and coumermycin clusters, respectively. The detection of cloJ and cloK in the clorobiocin cluster, and the homologous simJ1 and simK genes in the biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone (Galm et al., 2002 ; Trefzer et al., 2002
), now strongly supports the hypothesis that these genes are indeed related to aminocoumarin biosynthesis. cloJ shows homology to 3-oxo-acyl-[ACP] reductases and may likely be involved in the oxidation of a ß-hydroxy-tyrosyl to a ß-keto-tyrosyl intermediate (Fig. 3
). Also, cloK shows homology to oxidoreductases, but this homology is not very high (mean of 35% on the amino acid level). Chen & Walsh (2001)
speculated that NovJ and NovK may act together to oxidize the ß-hydroxyl function to a keto group. The unresolved step in the postulated Ring B biosynthesis, however, is the hydroxylation of the activated tyrosyl derivative in position 2 of the aromatic nucleus (Fig. 3
). Bunton et al. (1963)
had reported that the ring oxygen of the aminocoumarin may be derived from the carboxyl group of tyrosine rather than from molecular oxygen. This was recently disproven by Holzenkämpfer & Zeeck (2002)
, who showed that the ring oxygen of the aminocoumarin moiety of simocyclinone is in fact derived from molecular oxygen. Therefore, coumarin ring formation most likely proceeds via the 2-hydroxylation of a tyrosine derivative. Chen & Walsh (2001)
had speculated that the predicted flavine dioxygenase NovC, encoded by a gene near the novobiocin cluster, may catalyse this reaction. An important finding of our study is that no novC homologue was detected in or near the clorobiocin gene cluster. Likewise, no novC homologue was detected in the simocyclinone cluster (Galm et al., 2002
; Trefzer et al., 2002
). We therefore suggest that novC is not related to aminocoumarin biosynthesis. The enzyme responsible for the 2-hydroxylation of the ß-keto-tyrosyl intermediate remains unknown at present. Whether cloK is involved in this or another reaction of aminocoumarin biosynthesis has yet to be demonstrated.
Halogenation of the aminocoumarin ring may occur after ring formation, as depicted in Fig. 3, or at an earlier stage.
The prenylated 4-hydroxybenzoate moiety (Ring A) of clorobiocin and novobiocin is formed from tyrosine (Kominek & Sebek, 1974 ) and an isoprenoid precursor, but this reaction sequence is also unknown. The biosynthesis of this moiety requires: (i) the assembly of the isoprenoid precursor (probably dimethylallyl diphosphate) via the methylerythritol phosphate pathway (Li et al., 1998
); (ii) the conversion of the phenylpropanoid compound tyrosine to a benzoic acid derivative; and (iii) the prenylation of the aromatic nucleus in a prenyltransferase reaction. The conversion of the phenylpropanoid intermediate to a benzoic acid derivative may proceed by a mechanism analogous to the oxidation of fatty acids, as demonstrated in Streptomyces maritimus (Hertweck & Moore, 2000
). Alternatively, this conversion may occur by retro-aldol cleavage of a 3-hydroxylated phenylpropanoid compound, as found in P. fluorescens (Gasson et al., 1998
) and Amycolatopsis sp. (Achterholt et al., 2000
). Retro-aldol cleavage would result in a benzaldehyde derivate, which would subsequently be oxidized to the benzoic acid derivative.
In the clorobiocin cluster, we could not detect genes similar to those for the ß-oxidation of fatty acids. We did find, however, the gene cloR which showed homology to a putative aldolase from S. coelicolor. Comparison of the gene clusters of clorobiocin, novobiocin and coumermycin A1 led to the hypothesis that this gene may be involved in Ring A biosynthesis. Inactivation of the enzyme led to an abolishment of clorobiocin production. When the mutant was complemented with the prenylated 4-hydroxybenzoate moiety (=Ring A), clorobiocin production was restored. This proved that cloR is indeed involved in Ring A biosynthesis. We suggest that the formation of Ring A of clorobiocin may proceed via a retro-aldol reaction catalysed by CloR, i.e. by a mechanism different from the elucidated benzoic acid biosynthesis in S. maritimus (Hertweck & Moore, 2000 ).
The substrate of CloR may be an enzyme-bound prenylated ß-hydroxytyrosine, as suggested by Chen & Walsh (2001) and depicted in Fig. 3
. Alternatively, prenylated 4-hydroxyphenylpyruvate may be the substrate of CloR, since this compound was detected as the product of a prenyltransferase of the novobiocin producer S. spheroides (Steffensky et al., 1998
). Further studies are now in progress to investigate these steps in clorobiocin biosynthesis.
The cloning and sequencing of the clorobiocin gene cluster has completed the genetic information on the biosynthesis of three classical aminocoumarin antibiotics, namely novobiocin, clorobiocin and coumermycin A1. Comparison of the three gene clusters revealed a strikingly stringent correspondence between the structures of the antibiotics and the organization of the biosynthetic genes, unprecedented so far in any class of natural products outside the polyketide and the peptide antibiotics. For each structural moiety of the aminocoumarin antibiotics, the biosynthetic genes are grouped together, resulting in a modular structure of the clusters. The orders of the modules and the order of the genes within each module are perfectly identical for the three classical aminocoumarins, and nearly all of the genes within the clusters are orientated in the same direction. The comparison of the three clusters greatly facilitates the prediction of functions for the different genes. As an example, cloR was recognized by such a comparison as a candidate gene for the biosynthesis of the prenylated 4-hydroxybenzoate moiety of clorobiocin, and this was experimentally proven by an inactivation and complementation experiment. The similarity between the three clusters also provides excellent opportunities for the production of hybrid aminocoumarins by genetic methods.
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ACKNOWLEDGEMENTS |
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Received 28 May 2002;
revised 29 July 2002;
accepted 22 August 2002.