- Email: [email protected]
Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis
FEMS Microbiology Letters 170 (1999) 381^387
Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis Lucia Westrich a , Silvie Domann a , Bettina Faust a , David Bedford b , David A. Hopwood b , Andreas Bechthold a; * a
Institut fuër Pharmazeutische Biologie, Universitaët Tuëbingen, Auf der Morgenstelle 8, D-72076 Tuëbingen, Germany b John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received 4 September 1998; received in revised form 19 November 1998; accepted 22 November 1998
Abstract From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptomyces; Angucycline ; Landomycin; Biosynthetic gene cluster
1. Introduction Streptomyces cyanogenus S136 produces landomycins, consisting of a benz[a]anthraquinone-type aglycone moiety and a varying phenol-glycosidically linked oligosaccharide chain [1]. Landomycin A, the principal metabolite of S. cyanogenus, is the largest angucycline so far described, and the most active landomycin [2]. Its deoxysugar moiety is an unusual hexasaccharide consisting of four D-olivose and two L-rhodinose residues (Fig. 1). Over the past decade,
* Corresponding author. E-mail: [email protected]
the gene clusters encoding many structurally related aromatic polyketide synthases (PKSs) have been cloned and sequenced, among them the clusters for the angucycline antibiotics urdamycin A and jadomycin B [3^5]. All these studies paved the way for the construction of hybrid synthases by mixing PKS subunit genes from various parent microorganisms [6^8]. Many aromatic polyketides are modi¢ed by subsequent enzymes of the pathway, such as oxygenases and glycosyltransferases. These post-PKS tailoring enzymes are often responsible for converting inactive precursors into biologically active metabolites [9]. From investigations of the biosynthetic gene cluster for the landomycins, we expected to obtain interesting genes for combinatorial biosynthetic studies.
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2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions S. cyanogenus S136 (DSM 5087) was grown at 28³C on CRM medium for DNA isolation. Escherichia coli XLI-Blue MRF (Stratagene, La Jolla, CA, USA) was used for the preparation of the cosmid library and routine subcloning. pBluescript SK3 (pSK3 ) was obtained from Stratagene and cosmid pOJ446 was a gift from Eli Lilly Co. (Indianapolis, IN, USA). 2.2. DNA manipulation and southern hybridization
TDP-glucose, is one of the ¢rst enzymes involved in the biosynthesis of TDP-olivose and TDP-rhodinose [12]. Therefore approximately 2000 colonies containing recombinant cosmids were probed by colony hybridization using an internal fragment of a dNDP-glucose 4,6-dehydratase gene as a probe [15]. Cosmid DNA from seven hybridizing clones was analyzed by restriction mapping and Southern blot experiments. These revealed that the cosmids contained overlapping DNA encompassing approximately 70 kb of the S. cyanogenus S136 genome (Fig. 2). One cosmid, H2-26, was used for further experiments. 3.2. Sequence analysis of the putative landomycin biosynthetic gene cluster
Chromosomal DNA from S. cyanogenus S136 and plasmid DNA from E. coli were prepared using standard protocols [10,11]. A cosmid library was prepared as described [12]. Restriction endonuclease digestions, alkaline phosphatase treatments, ligation, Southern hybridization and other DNA manipulations were performed according to standard procedures as speci¢ed by the manufacturers (Amersham, Braunschweig; Boehringer, Mannheim).
CODONPREFERENCE analysis of 35-kb DNA revealed 30 open reading frames (ORFs). Two additional putative ORFs (lanU, lanZ6) were detected with a large number of codons with A or T in third position (Fig. 3, Table 1). The GenBank/EMBL/ IDDBJ accession number for the nucleotide sequence is AF080235.
2.3. DNA sequencing and computer assisted sequence analysis
3.3. Characterization of the deduced amino acid sequences
DNA was sequenced with an automated laser £uorescence sequencer (Vistra, Molecular Dynamics) according to the supplier's instructions using double-stranded DNA as template puri¢ed with Nucleobond AX-20 columns. Computer-assisted sequence analysis was carried out using the DNASIS software package (version 2, 1995; Hitachi Software Engineering, San Bruno, CA). BlastX analyses [13] were run with the GenBank CDC translations+PDB+SwissProt+Spupdate+PIR, release 2.0. Open reading frames were identi¢ed by using the CODONPREFERENCE program [14].
The deduced amino acid sequences encoded by lanA, lanB and lanC strongly resemble gene products of type II iterative polyketide synthases. The closest resemblance was found to UrdA (LanA: 81% identity), UrdB (LanB: 72%) and UrdC (LanC: 63%) from S. fradiae involved in the biosynthesis of urdamycins [3] and to Orf1 (LanA: 82% identity), Orf2 (LanB: 72%) and Orf3 (LanC: 70%) involved in jadomycin production [4]. The deduced LanD protein sequence has high similarity to ketoreductases from aromatic gene clusters that are involved in reduction of the C-9 keto group of the nascent polyketide chain. Again the highest resemblance was found to proteins of the urdamycin (UrdD, 85% identity) and jadomycin (ORF5, 86%) producers [3,4]. Comparison of the putative products of lanN and lanV to proteins in the data bases suggests that both belong to the short-chain alcohol dehydrogenase family of proteins. The highest resemblance was found to an ORF upstream of jadR2 in the jadomycin cluster [16]
3. Results and discussion 3.1. Screening of a cosmid library It was likely that a dTDP-glucose 4,6-dehydratase, catalyzing the deoxygenation reaction at C-6 of
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Fig. 1. Structure of landomycin A.
(LanN 40% identity; LanV 47%). Short-chain alcohol dehydrogenases, which are proteins of about 250 amino acids, exhibit a strictly conserved SX10^ 13YXXXK motif which is believed to form part of the active site. This motif is also present in LanN (SRGAVEPVEPKAAVYAMAK) and LanP (SSGLTRVASPDQVTYGMSK). The deduced protein products of two further genes (lanO and lanZ4) contain the motif VxVxPxxxxSxPxxxKxxxD which has been found in a NAD(P)H-dependent dehydrogenase from Bacillus subtilis (EMBL accession number: Z99121). The deduced amino acid sequence of lanP shows 53% identity to MmdA, a methylmalonyl-CoA decarboxylase from Veillonella parvula [17], and also a strong resemblance to several propionylCoA carboxylases from di¡erent organisms. All these proteins catalyze carboxyl transfer from methylmalonyl-CoA to protein-bound biotin to yield propionyl CoA and the carboxybiotin derivative, or catalyze carboxyl transfer in the reverse reaction. LanP might catalyze a decarboxylation step during the bio-
synthesis of the angucyclinone skeleton. The putative protein encoded by lanF is very similar to UrdF (73% identity) [3] and also similar to TcmI (38%) [18]. TcmI is believed to be involved in cyclization of the fourth ring during tetracenomycin biosynthesis. The deduced amino acid sequence of lanL resembles proteins designed as aromatases in di¡erent organisms. The closest resemblance was to JadORf4 (76% identity) [4], believed to catalyze aromatization of the ¢rst ring of jadomycin and actinorhodin respectively. The deduced amino acid sequences encoded by lanE and lanM strongly resemble proteins believed to be involved in oxygenation steps. Again, proteins from the urdamycin [3] and jadomycin [5] producers are most similar. Because the deduced amino acid sequence of lanZ5 shows signi¢cant similarity to ActVA-ORF5 [19], lanZ5 could be a third gene of the landomycin biosynthetic gene cluster encoding an oxygenase. The amino acid sequences encoded by lanG and lanZ2 are highly similar to GraD [12] (LanG: 69% identity, LanZ2: 46%) and StrD
Fig. 2. Restriction map of the cloned DNA from S. cyanogenus S136 DNA. Cosmid clones isolated are shown below the map.
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Fig. 3. Genetic organization of the putative landomycin biosynthetic gene cluster. Genes are indicated by arrows orientated in the direction of transcription (B, BamHI; Bg, BglII).
[20] (LanG: 52%, LanZ2: 42%), suggesting that they encode dNDP-hexose synthetases. The deduced amino acid sequence of LanH resembles several dehydratases (e.g., 65% identity to GraE from S. violaceoruber Tuë22 [12]) suggesting that lanH encodes a dTDP-glucose 4,6-dehydratase involved in C-6 dehydroxylation. LanQ strongly resembles AscC from Yersinia pseudotuberculosis (53% identity) [21] and proteins belonging to the SMAT (secondary metabolic aminotransferase) family [20]. AscC is involved in C-3 dehydroxylation. As the biosynthesis of L-rhodinose requires dehydroxylation at position C-3 LanQ might be involved in this reaction. The predicted LanS enzyme is 46% identical to DnmT from S. peucetius [22]. This enzyme may be involved as 2,3-dehydratases in deoxygenation at position C-2
of L-daunosamine. Amino acid residues 47^243 of LanS could be aligned to residues 262^461 (35% identity), indicating two similar substrate or cofactor binding sites in this protein. LanT is most related to RdmF from S. purpurascens [23] and a glucose-fructose oxidoreductase (Gfor) from Zymomonas mobilis [24]. The latter can catalyze oxidation of glucose to gluconolactone and the concomitant reduction of a fructose to sorbitol, during which the pyridine nucleotide cofactor remains bound to the protein. As a reduction and an oxidation step might be necessary for C-2 deoxygenation two enzymes (LanS and LanT) could be involved. LanR is most closely related to a UDP-glucose 4-epimerase (ExoB) from Azospirillum brasilense (31% identity) [25] and also shows some resemblance to a dTDP-6-deoxy-L-man-
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Table 1 Deduced functions of ORFs in the landomycin biosynthetic gene cluster ORF
Size of the protein (aa)
Proposed function
ORF
Size of the protein (aa)
Proposed function
lanE lanF lanA lanB lanC lanD lanL lanM lanN lanO lanP lanG lanH lanQ lanR lanS
491 109 424 406 89 261 319 522 222 192 524 355 326 434 253 469
oxygenase cyclase L-ketoacyl-ACP synthase chain length factor acyl carrier protein reductase cyclase oxygenase reductase reductase decarboxylase NDP-hexose synthetase NDP-hexose 4,6-dehydratase NDP-hexose 3,4-dehydratase NDP-hexose 4-ketoreductase NDP-hexose 2,3-dehydratase
lanT lanU lanV lanGT2 lanX lanGT1 lanK lanJ lanZ1 lanGT3 lanZ2 lanZ3 lanGT4 lanZ4 lanZ5 lanZ6
321 215 253 373 147 390 192 517 192 401 328 316 417 199 397 238
oxidoreductase ? reductase glycosyltransferase ? glycosyltransferase regulation transporter NDP-hexose 3,5-epimerase glycosyltransferase NDP-hexose sythetase NDP-hexose 4-ketoreductase glycosyltransferase reductase oxygenase ?
nose dehydrogenase (RhsD) from Sphingomonas strain S88 (28% identity) [26]. LanZ3 also resembles 4-ketoreductases. Here the highest resemblance was found to DnmV from S. peucetius (43% identity) involved in the biosynthesis of L-daunosamine as the sugar component of daunorubicin [27]. LanR and LanZ3 are probably involved in reducing 4-keto intermediates to NDP-D-olivose and NDP-Lrhodinose respectively. LanZ1 resembles DnmU from S. peucetius (45% identity) [27]. This enzyme is related to a large family of sequences that have been postulated to be involved in biosynthesis of L-sugars. LanGT1, LanGT2, LanGT3 and LanGT4 show signi¢cant similarity to putative glycosyltransferases [28], indicating an important function for these proteins in the biosynthesis of the sugar side chain. LanK resembles proteins involved in regulation of antibiotic biosynthesis. The closest similarity was found to JadR2 from S. venezuelae (29%) [16] and to TcmR from S. glaucescens (29%) [28,29]. Homologous regions were especially found in the Nterminal regions of these proteins. LanJ resembles hydrophobic proteins known as proton-dependent transporters of di¡erent drugs [30]. The closest resemblance was found to Brm3 from B. subtilis (EMBL accession number: D50098) and a hypothetical protein from Mycobacterium tuberculosis (EMBL accession number: Z97139). Interestingly all genes except lanK are transcribed in the same
direction. The fact that lanK, a putative regulatory gene, is transcribed divergently from lanJ provides the possibility that LanJ could be regulated by LanK at the level of transcription [31]. The function of two further genes in the cluster is completely unknown. LanX is similar to some proteins known to be B12-dependent. Resemblance was found to a methylmalonyl-CoA mutases (McmA2) from Archaeoglobus fulgidus (EMBL accession number: AE000952). LanZ6 resembles Nsh-ORFC, a protein with unknown function from S. actuosus [32]. No signi¢cant matches were found for LanU.
4. Conclusion We have sequenced a region of DNA from S. cyanogenus S136 spanning about 35 kb. The analysis of the DNA sequence is clearly consistent with the possibility that it could encode genes for the biosynthesis of landomycin but absolute proof was not possible, since all attempts to insertionally inactivate genes of the cluster through gene disruption or gene replacement failed. Introduction of isolated cosmids into S. lividans and S. coelicolor CH999 by transformation gave apramycin-resistant colonies, and some of these colonies produced compounds with a similar color and Rf value to some of the landomycins (TLC). However, because of the instability of the
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cosmids we could not produce enough material of these compounds necessary for NMR analysis. We also could not prove that the whole landomycin biosynthetic gene cluster has been cloned, but after sequencing an additional 0.7 kb on both sides (upstream of lanE and downstream of lanZ6) of the cluster no further putative related gene could be identi¢ed.
[9]
[10]
[11]
Acknowledgments We thank the Deutsche Forschungsgemeinschaft (SFB 323) and the European Community (BIO4CT96-0068) (grants to A.B.). Work at the John Innes Centre was supported by the John Innes Foundation and by the Biotechnology and Biological Science Research Council (grants to D.A.H.).
[12]
[13]
[14]
References [15] [1] Henkel, T., Rohr, J., Beale, J.M. and Schwenen, L. (1990) Landomycins. New angucycline antibiotics from Streptomyces sp. I. Structural studies on landomycins A^D. J. Antibiot. 43, 492^503. [2] Depenbrock, H., Bornschlegl, S., Peter, R., Rohr, J., Schmid, P., Schweighart, P., Block, T., Rastetter, J. and Hanauske, A.-R. (1996) Assessment of antitumor activity of landomycin A (NSC 6399187-A). Ann. Hematol. 73 (Suppl. II), A80/ 316. [3] Decker, H. and Haag, S. (1995) Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tuë2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation. J. Bacteriol. 177, 6126^6136. [4] Han, L., Yang, K., Ramalingam, E., Mosher, R.H. and Vining, L.C. (1994) Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230. Microbiology 140, 3379^3389. [5] Yang, K., Han, L., Ayer, S.W. and Vining, L.C. (1996) Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae. Microbiology 142, 123^132. [6] McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. and Khosla, C. (1993) Engineered biosynthesis of novel polyketides. Science 262, 1546^1550. [7] McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. and Khosla, C. (1995) Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375, 549^554. [8] Hutchinson, C.R. and Fujii, I. (1995) Polyketide synthase
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gene manipulation : a structure-function approach in engineering novel antibiotics. Annu. Rev. Microbiol. 49, 201^238. Motamedi, H., Sha¢ee, A., Cai, S.-J., Streicher, S.L., Arison, B.H. and Miller, R.R. (1996) Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of immunosuppressants FK506 and FK520. J. Bacteriol. 178, 5243^5248. Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schrempf, H. (1985) Genetic Manipulation of Streptomyces ^ A Laboratory Manual. The John Innes Foundation, Norwich. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Bechthold, A., Sohng, J.K., Smith, T.M., Chu, X. and Floss, H.G. (1995) Identi¢cation of Streptomyces violaceoruber Tuë22 genes involved in the biosynthesis of granaticin. Mol. Gen. Genet. 248, 610^620. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic alignment search tool. J. Mol. Biol. 215, 403^410. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 15, 387^395. Decker, H., Gaisser, S., Pelzer, S., Schneider, P., Westrich, L., Wohlleben, W. and Bechthold, A. (1996) A general approach for cloning and characterizing dNDP-glucose dehydratase genes from actinomycetes. FEMS Microbiol. Lett. 141, 195^ 201. Yang, K., Han, L. and Vining, L.C. (1995) Regulation of jadomycin production in Streptomyces venezuelae ISP5230: Involvement of a repressor gene, jadR2. J. Bacteriol. 177, 6111^6117. Huder, J.B. and Dimroth, P. (1993) Sequence of the sodium ion pump methylmalonyl-CoA decarboxylase from Veillonella parvula. J. Biol. Chem. 268, 24564^24571. Summers, R.G., Wendt-Pienkowski, E., Motamedi, H. and Hurchinson, C.R. (1993). The tcmVI region of the tetracenomycin C biosynthesis gene cluster of Streptomyces glaucescens encodes the teracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase. J. Bacteriol. 175, 7571^7580. Hopwood, D.A. (1997) Genetic contributions to understanding polyketide synthases. Chem. Rev. 97, 2466^2497. Piepersberg, W. (1994) Pathway engineering in secondary metabolite-producing actinomycetes. Crit. Rev. Biotechnol. 14, 251^285. Thorson, J.S., Lo, S.F. and Liu, H.-w. (1993) Molecular basis of 3,6-dideoxyhexose biosynthesis: elucidation of CDP-ascarylose biosynthetic genes and their relationship to other 3,6dideoxyhexose pathways. J. Am. Chem. Soc. 115, 5827^ 5828. Scotti, C. and Hutchinson, C.R. (1996) Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis. J. Bacteriol. 178, 7316^7321.
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L. Westrich et al. / FEMS Microbiology Letters 170 (1999) 381^387 [23] Niemi, J. and Maëntsaëlaë, P. (1995) Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus. J. Bacteriol. 177, 2942^2945. [24] Kanagasundaram, V. and Scopes, R.K. (1992) Cloning, sequence analysis, and expression of the structural gene encoding glucose-fructose oxidoreductase from Zymomonas mobilis. J. Bacteriol. 174, 1439^1447. [25] De Troch, P., Keijers, V. and Vanderleyden, J. (1994) Sequence analysis of the Azospirillum brasilense exoB gene, encoding UDP-glucose 4-epimerase. Gene 143^144. [26] Yamazaki, M., Thorne, L., Mikolajczak, M., Armentrout, R.W. and Pollock, T.J. (1996) Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88. J. Bacteriol. 178, 2676^2687. [27] Otten, S.L., Gallo, M.A., Madduri, K., Liu, X. and Hutchinson C.R. (1997) Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine. J. Bacteriol. 179, 4446^4450.
387
[28] Kirschning, A., Bechthold, A. and Rohr, J. (1997) Chemical and biochemical aspects of deoxysugars and therefrom derived oligosaccharides. Topics Curr. Chem. 188, 1^84. [29] Summers, R.G., Wendt-Pienkowski, E., Motamedi, H. and Hutchinson, C.R. (1992) Nucleotide sequence of the tcmIItcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase. J. Bacteriol. 174, 1810^1820. [30] Rouch, D.A., Cram, D.S., DiBeradino, D., Littlejohn, T.G. and Skurray, R.A. (1990) E¥ux mediated antiseptic resistance gene quacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol. Microbiol. 4, 2051^2062. [31] Otten, S.L., Ferguson, J. and Hutchinson, C.R. (1995) Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus. J. Bacteriol. 177, 1216^1224. [32] Li, Y., Dosch, D.C., Strohl, W.R. and Floss, H.G. (1990) Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene from Streptomyces actuosus. Gene 91, 9^17.
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Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis Lucia Westrich a , Silvie Domann a , Bettina Faust a , David Bedford b , David A. Hopwood b , Andreas Bechthold a; * a
Institut fuër Pharmazeutische Biologie, Universitaët Tuëbingen, Auf der Morgenstelle 8, D-72076 Tuëbingen, Germany b John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received 4 September 1998; received in revised form 19 November 1998; accepted 22 November 1998
Abstract From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Streptomyces; Angucycline ; Landomycin; Biosynthetic gene cluster
1. Introduction Streptomyces cyanogenus S136 produces landomycins, consisting of a benz[a]anthraquinone-type aglycone moiety and a varying phenol-glycosidically linked oligosaccharide chain [1]. Landomycin A, the principal metabolite of S. cyanogenus, is the largest angucycline so far described, and the most active landomycin [2]. Its deoxysugar moiety is an unusual hexasaccharide consisting of four D-olivose and two L-rhodinose residues (Fig. 1). Over the past decade,
* Corresponding author. E-mail: [email protected]
the gene clusters encoding many structurally related aromatic polyketide synthases (PKSs) have been cloned and sequenced, among them the clusters for the angucycline antibiotics urdamycin A and jadomycin B [3^5]. All these studies paved the way for the construction of hybrid synthases by mixing PKS subunit genes from various parent microorganisms [6^8]. Many aromatic polyketides are modi¢ed by subsequent enzymes of the pathway, such as oxygenases and glycosyltransferases. These post-PKS tailoring enzymes are often responsible for converting inactive precursors into biologically active metabolites [9]. From investigations of the biosynthetic gene cluster for the landomycins, we expected to obtain interesting genes for combinatorial biosynthetic studies.
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 6 1 - 8
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2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions S. cyanogenus S136 (DSM 5087) was grown at 28³C on CRM medium for DNA isolation. Escherichia coli XLI-Blue MRF (Stratagene, La Jolla, CA, USA) was used for the preparation of the cosmid library and routine subcloning. pBluescript SK3 (pSK3 ) was obtained from Stratagene and cosmid pOJ446 was a gift from Eli Lilly Co. (Indianapolis, IN, USA). 2.2. DNA manipulation and southern hybridization
TDP-glucose, is one of the ¢rst enzymes involved in the biosynthesis of TDP-olivose and TDP-rhodinose [12]. Therefore approximately 2000 colonies containing recombinant cosmids were probed by colony hybridization using an internal fragment of a dNDP-glucose 4,6-dehydratase gene as a probe [15]. Cosmid DNA from seven hybridizing clones was analyzed by restriction mapping and Southern blot experiments. These revealed that the cosmids contained overlapping DNA encompassing approximately 70 kb of the S. cyanogenus S136 genome (Fig. 2). One cosmid, H2-26, was used for further experiments. 3.2. Sequence analysis of the putative landomycin biosynthetic gene cluster
Chromosomal DNA from S. cyanogenus S136 and plasmid DNA from E. coli were prepared using standard protocols [10,11]. A cosmid library was prepared as described [12]. Restriction endonuclease digestions, alkaline phosphatase treatments, ligation, Southern hybridization and other DNA manipulations were performed according to standard procedures as speci¢ed by the manufacturers (Amersham, Braunschweig; Boehringer, Mannheim).
CODONPREFERENCE analysis of 35-kb DNA revealed 30 open reading frames (ORFs). Two additional putative ORFs (lanU, lanZ6) were detected with a large number of codons with A or T in third position (Fig. 3, Table 1). The GenBank/EMBL/ IDDBJ accession number for the nucleotide sequence is AF080235.
2.3. DNA sequencing and computer assisted sequence analysis
3.3. Characterization of the deduced amino acid sequences
DNA was sequenced with an automated laser £uorescence sequencer (Vistra, Molecular Dynamics) according to the supplier's instructions using double-stranded DNA as template puri¢ed with Nucleobond AX-20 columns. Computer-assisted sequence analysis was carried out using the DNASIS software package (version 2, 1995; Hitachi Software Engineering, San Bruno, CA). BlastX analyses [13] were run with the GenBank CDC translations+PDB+SwissProt+Spupdate+PIR, release 2.0. Open reading frames were identi¢ed by using the CODONPREFERENCE program [14].
The deduced amino acid sequences encoded by lanA, lanB and lanC strongly resemble gene products of type II iterative polyketide synthases. The closest resemblance was found to UrdA (LanA: 81% identity), UrdB (LanB: 72%) and UrdC (LanC: 63%) from S. fradiae involved in the biosynthesis of urdamycins [3] and to Orf1 (LanA: 82% identity), Orf2 (LanB: 72%) and Orf3 (LanC: 70%) involved in jadomycin production [4]. The deduced LanD protein sequence has high similarity to ketoreductases from aromatic gene clusters that are involved in reduction of the C-9 keto group of the nascent polyketide chain. Again the highest resemblance was found to proteins of the urdamycin (UrdD, 85% identity) and jadomycin (ORF5, 86%) producers [3,4]. Comparison of the putative products of lanN and lanV to proteins in the data bases suggests that both belong to the short-chain alcohol dehydrogenase family of proteins. The highest resemblance was found to an ORF upstream of jadR2 in the jadomycin cluster [16]
3. Results and discussion 3.1. Screening of a cosmid library It was likely that a dTDP-glucose 4,6-dehydratase, catalyzing the deoxygenation reaction at C-6 of
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Fig. 1. Structure of landomycin A.
(LanN 40% identity; LanV 47%). Short-chain alcohol dehydrogenases, which are proteins of about 250 amino acids, exhibit a strictly conserved SX10^ 13YXXXK motif which is believed to form part of the active site. This motif is also present in LanN (SRGAVEPVEPKAAVYAMAK) and LanP (SSGLTRVASPDQVTYGMSK). The deduced protein products of two further genes (lanO and lanZ4) contain the motif VxVxPxxxxSxPxxxKxxxD which has been found in a NAD(P)H-dependent dehydrogenase from Bacillus subtilis (EMBL accession number: Z99121). The deduced amino acid sequence of lanP shows 53% identity to MmdA, a methylmalonyl-CoA decarboxylase from Veillonella parvula [17], and also a strong resemblance to several propionylCoA carboxylases from di¡erent organisms. All these proteins catalyze carboxyl transfer from methylmalonyl-CoA to protein-bound biotin to yield propionyl CoA and the carboxybiotin derivative, or catalyze carboxyl transfer in the reverse reaction. LanP might catalyze a decarboxylation step during the bio-
synthesis of the angucyclinone skeleton. The putative protein encoded by lanF is very similar to UrdF (73% identity) [3] and also similar to TcmI (38%) [18]. TcmI is believed to be involved in cyclization of the fourth ring during tetracenomycin biosynthesis. The deduced amino acid sequence of lanL resembles proteins designed as aromatases in di¡erent organisms. The closest resemblance was to JadORf4 (76% identity) [4], believed to catalyze aromatization of the ¢rst ring of jadomycin and actinorhodin respectively. The deduced amino acid sequences encoded by lanE and lanM strongly resemble proteins believed to be involved in oxygenation steps. Again, proteins from the urdamycin [3] and jadomycin [5] producers are most similar. Because the deduced amino acid sequence of lanZ5 shows signi¢cant similarity to ActVA-ORF5 [19], lanZ5 could be a third gene of the landomycin biosynthetic gene cluster encoding an oxygenase. The amino acid sequences encoded by lanG and lanZ2 are highly similar to GraD [12] (LanG: 69% identity, LanZ2: 46%) and StrD
Fig. 2. Restriction map of the cloned DNA from S. cyanogenus S136 DNA. Cosmid clones isolated are shown below the map.
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Fig. 3. Genetic organization of the putative landomycin biosynthetic gene cluster. Genes are indicated by arrows orientated in the direction of transcription (B, BamHI; Bg, BglII).
[20] (LanG: 52%, LanZ2: 42%), suggesting that they encode dNDP-hexose synthetases. The deduced amino acid sequence of LanH resembles several dehydratases (e.g., 65% identity to GraE from S. violaceoruber Tuë22 [12]) suggesting that lanH encodes a dTDP-glucose 4,6-dehydratase involved in C-6 dehydroxylation. LanQ strongly resembles AscC from Yersinia pseudotuberculosis (53% identity) [21] and proteins belonging to the SMAT (secondary metabolic aminotransferase) family [20]. AscC is involved in C-3 dehydroxylation. As the biosynthesis of L-rhodinose requires dehydroxylation at position C-3 LanQ might be involved in this reaction. The predicted LanS enzyme is 46% identical to DnmT from S. peucetius [22]. This enzyme may be involved as 2,3-dehydratases in deoxygenation at position C-2
of L-daunosamine. Amino acid residues 47^243 of LanS could be aligned to residues 262^461 (35% identity), indicating two similar substrate or cofactor binding sites in this protein. LanT is most related to RdmF from S. purpurascens [23] and a glucose-fructose oxidoreductase (Gfor) from Zymomonas mobilis [24]. The latter can catalyze oxidation of glucose to gluconolactone and the concomitant reduction of a fructose to sorbitol, during which the pyridine nucleotide cofactor remains bound to the protein. As a reduction and an oxidation step might be necessary for C-2 deoxygenation two enzymes (LanS and LanT) could be involved. LanR is most closely related to a UDP-glucose 4-epimerase (ExoB) from Azospirillum brasilense (31% identity) [25] and also shows some resemblance to a dTDP-6-deoxy-L-man-
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Table 1 Deduced functions of ORFs in the landomycin biosynthetic gene cluster ORF
Size of the protein (aa)
Proposed function
ORF
Size of the protein (aa)
Proposed function
lanE lanF lanA lanB lanC lanD lanL lanM lanN lanO lanP lanG lanH lanQ lanR lanS
491 109 424 406 89 261 319 522 222 192 524 355 326 434 253 469
oxygenase cyclase L-ketoacyl-ACP synthase chain length factor acyl carrier protein reductase cyclase oxygenase reductase reductase decarboxylase NDP-hexose synthetase NDP-hexose 4,6-dehydratase NDP-hexose 3,4-dehydratase NDP-hexose 4-ketoreductase NDP-hexose 2,3-dehydratase
lanT lanU lanV lanGT2 lanX lanGT1 lanK lanJ lanZ1 lanGT3 lanZ2 lanZ3 lanGT4 lanZ4 lanZ5 lanZ6
321 215 253 373 147 390 192 517 192 401 328 316 417 199 397 238
oxidoreductase ? reductase glycosyltransferase ? glycosyltransferase regulation transporter NDP-hexose 3,5-epimerase glycosyltransferase NDP-hexose sythetase NDP-hexose 4-ketoreductase glycosyltransferase reductase oxygenase ?
nose dehydrogenase (RhsD) from Sphingomonas strain S88 (28% identity) [26]. LanZ3 also resembles 4-ketoreductases. Here the highest resemblance was found to DnmV from S. peucetius (43% identity) involved in the biosynthesis of L-daunosamine as the sugar component of daunorubicin [27]. LanR and LanZ3 are probably involved in reducing 4-keto intermediates to NDP-D-olivose and NDP-Lrhodinose respectively. LanZ1 resembles DnmU from S. peucetius (45% identity) [27]. This enzyme is related to a large family of sequences that have been postulated to be involved in biosynthesis of L-sugars. LanGT1, LanGT2, LanGT3 and LanGT4 show signi¢cant similarity to putative glycosyltransferases [28], indicating an important function for these proteins in the biosynthesis of the sugar side chain. LanK resembles proteins involved in regulation of antibiotic biosynthesis. The closest similarity was found to JadR2 from S. venezuelae (29%) [16] and to TcmR from S. glaucescens (29%) [28,29]. Homologous regions were especially found in the Nterminal regions of these proteins. LanJ resembles hydrophobic proteins known as proton-dependent transporters of di¡erent drugs [30]. The closest resemblance was found to Brm3 from B. subtilis (EMBL accession number: D50098) and a hypothetical protein from Mycobacterium tuberculosis (EMBL accession number: Z97139). Interestingly all genes except lanK are transcribed in the same
direction. The fact that lanK, a putative regulatory gene, is transcribed divergently from lanJ provides the possibility that LanJ could be regulated by LanK at the level of transcription [31]. The function of two further genes in the cluster is completely unknown. LanX is similar to some proteins known to be B12-dependent. Resemblance was found to a methylmalonyl-CoA mutases (McmA2) from Archaeoglobus fulgidus (EMBL accession number: AE000952). LanZ6 resembles Nsh-ORFC, a protein with unknown function from S. actuosus [32]. No signi¢cant matches were found for LanU.
4. Conclusion We have sequenced a region of DNA from S. cyanogenus S136 spanning about 35 kb. The analysis of the DNA sequence is clearly consistent with the possibility that it could encode genes for the biosynthesis of landomycin but absolute proof was not possible, since all attempts to insertionally inactivate genes of the cluster through gene disruption or gene replacement failed. Introduction of isolated cosmids into S. lividans and S. coelicolor CH999 by transformation gave apramycin-resistant colonies, and some of these colonies produced compounds with a similar color and Rf value to some of the landomycins (TLC). However, because of the instability of the
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cosmids we could not produce enough material of these compounds necessary for NMR analysis. We also could not prove that the whole landomycin biosynthetic gene cluster has been cloned, but after sequencing an additional 0.7 kb on both sides (upstream of lanE and downstream of lanZ6) of the cluster no further putative related gene could be identi¢ed.
[9]
[10]
[11]
Acknowledgments We thank the Deutsche Forschungsgemeinschaft (SFB 323) and the European Community (BIO4CT96-0068) (grants to A.B.). Work at the John Innes Centre was supported by the John Innes Foundation and by the Biotechnology and Biological Science Research Council (grants to D.A.H.).
[12]
[13]
[14]
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