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Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H

2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês

10.1074/jbc.m807534200

1083-351X

Wenqing Chen, Tingting Huang, Xinyi He, Qingqing Meng, Delin You, Linquan Bai, Jialiang Li, Mingxuan Wu, Rui Li, Zhoujie Xie, Huchen Zhou, Xiufen Zhou, Huarong Tan, Zixin Deng,

Seaweed-derived Bioactive Compounds

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics. A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics. Nucleoside antibiotics are a family of important microbial secondary metabolites with a wide range of bioactive properties. They originate by a combination of several primary metabolic pathways, including those of nucleic acids, proteins, and glycans (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar). Polyoxins (Fig. 1A), a group of structurally related nucleoside antibiotics produced by Streptomyces cacaoi var. asoensis (S. cacaoi hereafter) (2Isono K. Nagatsu J. Kobinata K. Sazuki K. Suzuki S. Agric. Biol. Chem... 1965; 29: 848-854Google Scholar, 3Isono K.N. Kobinata K. Sasaki K. Suzuki S. Agric. Biol. Chem... 1967; 31: 190-199Google Scholar) and Streptomyces aureochromogenes (4Wen Z. Chinese J. Biol... 2004; 21: 36-37Google Scholar), exhibit powerful bioactivity against phytopathogenic fungi (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar, 5Hori M. Eguchi J. Kakiki K. Misato T. J. Antibiot. (Tokyo).. 1974; 27: 260-266Google Scholar). As the first discovered nucleoside antibiotic inhibiting fungal cell wall biosynthesis, polyoxin was known to act as a competitive inhibitor of the chitin synthetase (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar, 5Hori M. Eguchi J. Kakiki K. Misato T. J. Antibiot. (Tokyo).. 1974; 27: 260-266Google Scholar) because of its intrinsic structural mimic of UDP-N-acetylglucosamine, a substrate for chitin biosynthesis. Polyoxin has played an outstanding role as an efficient agricultural fungicide without unwanted toxicity ever since its discovery in 1965 (6Zhang D. Miller M.J. Curr. Pharm. Des... 1999; 5: 73-99Google Scholar). Polyoxin was composed of three moieties (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar), including a nucleoside skeleton and two modified amino acids, polyoximic acid (POIA) 3The abbreviations used are: POIA, polyoximic acid; PEP, phosphoenolpyruvate; 3′-EUMP, 3′-enolpyruvyl-UMP; IPTG, isopropyl β-d-thiogalactopyranoside; CPOAA, carbamoylpolyoxamic acid; AHV, α-amino-δ-hydroxyvaleric acid; ACV, α-amino-δ-carbamoylhydroxyvaleric acid; LC/MS, liquid chromatography/mass spectrometry; MS, mass spectrometry; MS/MS, tandem MS; HPLC, high pressure liquid chromatography. and carbamoylpolyoxamic acid (CPOAA) (2Isono K. Nagatsu J. Kobinata K. Sazuki K. Suzuki S. Agric. Biol. Chem... 1965; 29: 848-854Google Scholar, 3Isono K.N. Kobinata K. Sasaki K. Suzuki S. Agric. Biol. Chem... 1967; 31: 190-199Google Scholar, 6Zhang D. Miller M.J. Curr. Pharm. Des... 1999; 5: 73-99Google Scholar). Isotope feeding experiments demonstrated that the nucleoside skeleton was initiated using uridine and phosphoenolpyruvate (PEP) as substrates, and the two modified amino acids originated from l-isoleucine and l-glutamate (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar) independently. Reports on the cloning of biosynthetic gene clusters for nucleoside antibiotics were limited to complete pathways for a purine nucleoside antibiotic puromycin (7Lacalle R.A. Tercero J.A. Jimenez A. EMBO J... 1992; 11: 785-792Google Scholar), a pyrimidine nucleoside antibiotic nikkomycin, as well as a partial pyrimidine nucleoside antibiotic blasticidin S (8Cone M.C. Yin X. Grochowski L.L. Parker M.R. Zabriskie T.M. ChemBioChem.. 2003; 4: 821-828Google Scholar). Whereas the puromycin biosynthetic pathway was completely demonstrated, the biosynthetic mechanism for blasticidin S was incompletely understood. A tentative biosynthetic pathway for nikkomycin Z (Fig. 1B), produced by Streptomyces tendae Tü901 (1Isono K. J. Antibiot. (Tokyo).. 1988; 41: 1711-1739Google Scholar) and Streptomyces ansochromogenes (9Liu G. Tian Y. Yang H. Tan H. Mol. Microbiol... 2005; 55: 1855-1866Google Scholar), has been elucidated (10Bruntner C. Bormann C. Eur. J. Biochem... 1998; 254: 347-355Google Scholar, 11Bruntner C. Lauer B. Schwarz W. Mohrle V. Bormann C. Mol. Gen. Genet... 1999; 262: 102-114Google Scholar, 12Lauer B. Russwurm R. Bormann C. Eur. J. Biochem... 2000; 267: 1698-1706Google Scholar, 13Lauer B. Sussmuth R. Kaiser D. Jung G. Bormann C. J. Antibiot. (Tokyo).. 2000; 53: 385-392Google Scholar, 14Lauer B. Russwurm R. Schwarz W. Kalmanczhelyi A. Bruntner C. Rosemeier A. Bormann C. Mol. Gen. Genet... 2001; 264: 662-673Google Scholar, 15Chen H. Hubbard B.K. O'Connor S.E. Walsh C.T. Chem. Biol... 2002; 9: 103-112Google Scholar, 16Ginj C. Ruegger H. Amrhein N. Macheroux P. ChemBioChem.. 2005; 6: 1974-1976Google Scholar-17Carrell C.J. Bruckner R.C. Venci D. Zhao G. Jorns M.S. Mathews F.S. Structure (Lond.).. 2007; 15: 928-941Google Scholar) to derive from two moieties involving a nucleoside skeleton and a modified amino acid originating from l-tyrosine, but the precise mechanism for the biosynthesis of its nucleoside skeleton, which seemed similar to polyoxin, remains to be clarified (16Ginj C. Ruegger H. Amrhein N. Macheroux P. ChemBioChem.. 2005; 6: 1974-1976Google Scholar). NikO, a key enzyme essential for biosynthesis of the nikkomycin nucleoside skeleton, was recently demonstrated to use UMP, instead of uridine, with PEP to form a novel and unexpected product 3′-enolpyruvyl-UMP (3′-EUMP) (16Ginj C. Ruegger H. Amrhein N. Macheroux P. ChemBioChem.. 2005; 6: 1974-1976Google Scholar). This is totally different from the previously proposed pathway for the biosynthesis of the polyoxin nucleoside skeleton. Here we describe the cloning and functional analysis of a complete polyoxin biosynthetic gene cluster. The heterologous production of polyoxin H in a nonproducer, Streptomyces lividans TK24, helped us to pinpoint an essential region consisting of 20 putative pol genes of 39 predicted open reading frames in a sequenced region as large as 46 kb, which led to a proposal of putative pathway for polyoxin biosynthesis. The availability of these genes will significantly help the elucidation of the exact biosynthetic mechanism of polyoxin, and for the more rational generation of polyoxin derivatives with novel or enhanced bioactivities via strategies involving pathway engineering or combinatorial biosynthesis. Bacterial Strains, Plasmids (Cosmids), General Methods, and Culture Conditions—Bacterial strains and plasmids (cosmids) used are described in supplemental Table S1. S. cacaoi and its derivatives were grown on MS agar (18Kieser T. Bibb M.J. Chater K.F. Butter M.J. Hopwood D.A. Practical Streptomyces Genetics, 2nd Ed..John Innes Foundation, Norwich, UK. 2000; : 169-170Google Scholar) or in TSB liquid medium (18Kieser T. Bibb M.J. Chater K.F. Butter M.J. Hopwood D.A. Practical Streptomyces Genetics, 2nd Ed..John Innes Foundation, Norwich, UK. 2000; : 169-170Google Scholar) at 30 °C. Liquid fermentation medium (containing the following per liter: 20 g of soy powder, 15 g of corn powder, 10 g of glucose, 10 g of yeast extract, 4 g of CaCO3, 2 g of KH2PO4, 2 g of NaCl) was used for polyoxin production. General approaches for Escherichia coli or Streptomyces manipulation were according to the standard methods of Sambrook et al. (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed..Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989; Google Scholar) or Kieser et al. (18Kieser T. Bibb M.J. Chater K.F. Butter M.J. Hopwood D.A. Practical Streptomyces Genetics, 2nd Ed..John Innes Foundation, Norwich, UK. 2000; : 169-170Google Scholar). The final antibiotic concentrations used for selection of E. coli or Streptomyces were as follows: 100 μg/ml ampicillin, 30 μg/ml apramycin, 50 μg/ml kanamycin, and 12.5 μg/ml thiostrepton. Milli-Q water purified by Milli-Q® ultrapure water purification systems was used throughout except for medium preparation. PCR Primers, DNA Probes, and Southern Blot—PCR primers used are listed in supplemental Table S2. For Southern blot experiments, S. cacaoi genomic DNA was digested with specific restriction enzymes, separated on 0.7% agarose gels overnight, and transferred onto Hybond-N+ nylon membrane (Amersham Biosciences). With use of [α-32P]dCTP-labeled radioactive probes by the random priming kit (Takara), Southern blot experiments were performed based on the standard protocol by Sambrook et al. (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed..Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989; Google Scholar). Genomic Library Construction for S. cacaoi and the Strategy for Screening Positive Cosmids—For construction of the pOJ446-derived genomic library for S. cacaoi, the standard method (18Kieser T. Bibb M.J. Chater K.F. Butter M.J. Hopwood D.A. Practical Streptomyces Genetics, 2nd Ed..John Innes Foundation, Norwich, UK. 2000; : 169-170Google Scholar, 19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed..Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989; Google Scholar) was adopted, and the competent EPI300-T1R E. coli was selected as host. For screening the cosmid genomic library by PCR, the primers (PolAF and PolAR) were used, and cosmid (mix) was used as template. The positive clones were identified in 96-well plates. Nucleotide Sequence Accession Number—The nucleotide sequence reported in this paper is available in the GenBank™ data base under accession number EU158805. DNA Sequencing and Sequence Analysis—DNA sequencing was accomplished at Invitrogen using pIJ2925 (18Kieser T. Bibb M.J. Chater K.F. Butter M.J. Hopwood D.A. Practical Streptomyces Genetics, 2nd Ed..John Innes Foundation, Norwich, UK. 2000; : 169-170Google Scholar) as vector, and sequencing reactions were carried out in an Applied Biosystems model 3730 automated DNA sequencer. Sequence data analysis was performed with FramePlot 3.0beta online program (20Ishikawa J. Hotta K. FEMS Microbiol. Lett... 1999; 174: 251-253Google Scholar). Nucleotide and amino acid sequence homology searches were performed using BLAST (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol... 1990; 215: 403-410Google Scholar). Heterologous Production of Polyoxin H—For heterologous production of polyoxin in S. lividans TK24, an EcoRI-XbaI engineered fragment bearing the int gene and the attp site from pSET152 was amplified by PCR with primers 5a7mF and 5a7mR, and this engineered fragment was used to replace the corresponding region in cosmid 5A7 containing the unstable SCP2 replicon to generate the modified 5A7 (m5A7), which was introduced into S. lividans TK24 for the engineered production of polyoxin. Construction and Identification of a polA Mutant—For construction of a polA targeted disruption vector, an ∼3.0-kb PvuII fragment harboring polA from cosmid 8B9 was cloned into the SmaI site of pIJ2925 to generate pJTU2158. With the primers Podf and Podr carrying XbaI and EcoRI site individually, the ∼3.0-kb fragment was amplified by KOD-plus DNA polymerase (Toyobo) using pJTU2158 as template; subsequently, this XbaI-EcoRI engineered fragment was cloned into pJTU1278, 4H. E. Yunlong, unpublished data. a derivative of pHZ1358 (22Sun Y. Zhou X. Liu J. Bao K. Zhang G. Tu G. Kieser T. Deng Z. Microbiology.. 2002; 148: 361-371Google Scholar), to give pJTU2161, and a BamHI fragment containing aac(3)IV (apramycin resistance gene) from pHZ1070 (23Li W. Ying X. Guo Y. Yu Z. Zhou X. Deng Z. Kieser H. Chater K.F. Tao M. J. Bacteriol... 2006; 188: 8368-8375Google Scholar) was inserted into the internal counterpart site of polA in pJTU2161 to produce the polA targeted disruption vector pJTU2165. Both PCR and Southern blot experiments were carried out to identify the CY1 mutants. For PCR identification, the primers PiomF and PiomR were used. The additional Southern blot validation used an ∼0.7-kb polA fragment obtained from pJTU2152 as a probe. Complementation of a Mutant CY1 with nikO and polA—With primers NikO-EXF and NikO-EXR, the complete nikO structural gene (with start codon GTG changed to ATG) was amplified with KOD-plus polymerase, treated with EcoRI, and then cloned into pIJ2925 (cleaved with EcoRI and SmaI) to generate pJTU2173. With primers polAef and polAer, the polA structural gene (with start codon GTG changed to ATG) was amplified, cut with EcoRI, and inserted into pBluescript II (SK+) (digested with EcoRI and SmaI) to produce pJTU2810. Two sequencing-confirmed EcoRI-NdeI fragments harboring the structural genes nikO from pJTU2173 and polA from pJTU2810, respectively, were cloned into the corresponding sites of pJTU695 (24Bai L. Li L. Xu H. Minagawa K. Yu Y. Zhang Y. Zhou X. Floss H.G. Mahmud T. Deng Z. Chem. Biol... 2006; 13: 387-397Google Scholar) to form pJTU2179 and pJTU2198. Construction and Identification of a polO Mutant—A 4.1-kb PvuII fragment from cosmid 5A7 containing intact polO was cloned into pBlueScriptII SK(+) to generate pJTU2900, from which a HindIII-SpeI fragment was cloned into the HindIII-XbaI site of pJTU1289 4H. E. Yunlong, unpublished data. to give pJTU2940, and an aac(3)IV+oriT cassette was recombined into the pJTU2940 by PCR targeting technology (25Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A... 2003; 100: 1541-1546Google Scholar) to produce pJTU2941 as a vector for polO disruption. Primers (polOIDF and polOIDR) were used for the identification of polO mutant. Polyoxin Purification and Assay—Polyoxin produced by S. cacaoi var. asoensis and its derivatives was detected by bioassay, HPLC (Waters 220), and LC/MS (Agilent 1100 series LC/MSD Trap system). For the bioassay, Trichosporon cutaneum was used as indicator strain. For HPLC and LC/MS analysis, the prepared broth containing polyoxin was purified by Dowex 50W×8(H+) resin, and the targeted fraction was collected and condensed before HPLC and LC/MS analysis. The conditions for HPLC analysis were according to the method of Fiedler (26Fiedler H.P. J. Chromatogr... 1984; 316: 487-494Google Scholar) except that the flow rate was 0.5 ml/min. The elution was monitored at 263.6 nm with a PDA (DAD) detector, and the data were analyzed with Waters Millennium Chromatography Manager (Agilent data analysis software). Quantification of Polyoxin H/A—For quantification of polyoxin H/A, polyoxin H/A standards were independently prepared by Shimadzu LC-8A preparative liquid chromatography. The HPLC conditions were as follows: 0.15% trifluoroacetic acid was increased to 50% in 30 min, and acetonitrile was correspondingly decreased to 50%; flow rate was 5 ml/min; after that, the standard curve was drawn according HPLC peak area of series polyoxin H/A concentrations (50, 100, 200, and 500 μg/ml, respectively) (see above for HPLC under "Polyoxin Purification and Assay") before the quantity of the polyoxin H/A in crude broth was calculated. Conditions for MS Analysis—The ion trap mass spectrometer (Agilent 1100 series LC/MSD trap system was operated with the electrospray ionization source) analysis for polyoxin and ACV (AHV) was in positive mode and for 3′-EUMP (UMP) in negative mode. The parameters for all MS analysis are as follows: drying gas flow was 10 liters/ml, and nebulizer pressure was 30 p.s.i.; drying gas temperature was 325 °C. Heterologous Overexpression of Recombinant His6-tagged PolA, NikO, and PolO—For heterologous expression of PolA, NikO, and PolO in E. coli BL21(DE3)/pLysE (Stratagene), both EcoRI-NdeI fragments harboring the structural genes nikO from pJTU2173, polA from pJTU2810, and polO from pJTU2896 were individually cloned into the corresponding sites of pET28a (Stratagene) to obtain pJTU2178, pJTU2197, and pET28a/polO. The plasmid DNA was transformed into E. coli BL21 (DE3)/pLysE. The transformants were grown in 600 ml of LB medium containing kanamycin and chloramphenicol at 37 °C to A600 of 0.6 before addition of isopropyl β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mm, and the incubation was continued at 30 °C for 3 h. The cells were then harvested by centrifugation at 6000 rpm for 10 min, and the pellet resuspended in 60 ml of binding buffer (150 mm NaCl, 20 mm Tris-HCl, pH 7.5) was stored frozen at -70 °C until further use. Preparation of Cell-free Extracts and Purification of His6-tagged PolA, NikO, and PolO—After thawing in water, the E. coli cells were lysed by sonication, and the cell debris was removed by centrifugation at 15,000 rpm for at least 30 min. Recombinant His-tagged protein was purified from the supernatant by gravity-flow chromatography on nickel-nitrilotriacetic acid-agarose (Qiagen). The bound recombinant protein was then eluted off the nickel column with gradient elution buffer (500 mm imidazole, 150 mm NaCl, 20 mm Tris-HCl, pH 7.5) at a flow rate 0.5 ml/min by Amersham Biosciences ÄKTA FPLC, and glycerol was added to the purified protein to a final concentration of 10% to prevent precipitation. After that, the purified recombinant protein was aliquoted and stored at -70 °C. In Vitro Enzyme Assay for PolA and NikO—In vitro characterization of PolA and NikO was according to the method of Ginj et al. (16Ginj C. Ruegger H. Amrhein N. Macheroux P. ChemBioChem.. 2005; 6: 1974-1976Google Scholar). The LC/MS analysis conditions used to judge the enzyme-catalyzed product were as follows: 91% of 0.15% trifluoroacetic acid, 9% of methanol, flow rate 0.3 ml/min. Compounds were analyzed by recording the absorbancy at 260 nm (for UMP and 3′-EUMP). Before injection of samples into LC/MS, protein was precipitated by adding an equal volume of 14% trichloroacetic acid to the reaction mix. In Vitro Enzyme Assay for PolO—In vitro enzyme assay of PolO was carried out at 30 °C overnight (under the following conditions: 200 μl volume containing phosphate buffer, pH 7.2, 50 mm, AHV 1 mm, MgSO4 5 mm, ATP 2 mm, dithiothreitol 2 mm, carbamoylphosphate 1 mm, PolO 10 μm). Before LC/MS analysis, protein was precipitated using the conditions described above, and the LC/MS analysis conditions used to judge the enzyme-catalyzed product were as follows: 90% of 0.15% trifluoroacetic acid, 10% of acetonitrile, flow rate 0.3 ml/min. The procedure for TLC analysis of PolO-catalyzed product was as follows: PolO-catalyzed product was initially purified by Dowex 50W×8(H+), separated by TLC with the solvent system butanol/acetic acid/water (4:1:2), and visualized by ninhydrin solution (1.5 g of ninhydrin dissolved in 100 ml of butanol and 3 ml of acetic acid). Synthesis of AHV—Synthesis of AHV was according to method of Garcia et al. (27Garcia M. Serra A. Rubiralta M. Diez A. Segarra V. Lozoya E. Ryder H. Palacios J.M. Tetrahedron Asymmetry.. 2000; 11: 991-994Google Scholar). The synthesized AHV was confirmed by 1H NMR (D2O, 400 MHz) δ 3.58 (t, J = 5.6 Hz, 1H), 3.45 (t, J = 6.4 Hz, 2H), 1.70–1.77 (m, 2H), 1.38–1.51 (m, 2H). Genetic Organization of the Cloned Polyoxin Gene Cluster—When an ∼0.7-kb nikO fragment from S. tendae Tü901 for the nikkomycin biosynthesis was amplified with primers NikOTF and NikOTR for use to probe the BamHI-digested genomic DNA of the two polyoxin producers (S. cacaoi var. asoensis and S. aureochromogenes (4Wen Z. Chinese J. Biol... 2004; 21: 36-37Google Scholar)) at low stringency, weak hybridization signals at ∼1.6 and ∼3.5 kb, respectively (see supplemental Fig. S1), appeared, which indicates that both strains may contain nikO homologs. To isolate a nikO homolog as a probe for screening cosmids from a genomic library of S. cacaoi, a distinct PCR product of 723 bp, deduced to encode a 240-amino acid NikO homolog, was first amplified from S. aureochromogenes using specific primers NikOTF and NikOTR, from which another pair of primers (PolAf and PolAr) was designed for the amplification of a 549-bp PCR product simultaneously from S. cacaoi and S. aureochromogenes. Both PCR products showed 99% identity within the shared 549-bp region. A pOJ446-derived cosmid library was screened by PCR (see "Experimental Procedures"). Of ∼2,000 cosmids, 8 gave positive signals, which spanned ∼65-kb contiguous region of S. cacaoi as revealed by mapping with PvuII (Fig. 2A). A contiguous 46,066-bp sequenced region arising from sequencing one of the centrally located cosmids 5A7 (∼35 kb), and subsequently an additional XhoI (4.8 kb) and two internal PvuII (1.5 and 6.7 kb) fragments (GenBank™ accession number EU158805, Fig. 2A), had an overall G + C content of 72.58%, similar to that of a typical Streptomyces genome exemplified by Streptomyces coelicolor A3(2) (28Bentley S.D. Chater K.F. Cerdeno-Tarraga A.M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbinowitsch E. Rajandream M.A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature.. 2002; 417: 141-147Google Scholar) and Streptomyces avermitilis (29Omura S. Ikeda H. Ishikawa J. Hanamoto A. Takahashi C. Shinose M. Takahashi Y. Horikawa H. Nakazawa H. Osonoe T. Kikuchi H. Shiba T. Sakaki Y. Hattori M. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 12215-12220Google Scholar). The frame plot 3.0 beta online program revealed 39 complete open reading frames whose organization is shown in Fig. 2B, with putative functions described in Table 1.Table 1Deduced functions of the open reading frames in the pol gene cluster Protein Amino acids Proposed function Homolog, origin Identity, similarity Accession no. % PolB 257 Thymidylate synthase SCO5743, S. coelicolor A3(2) 60, 72 NP_629868 PolAaFunctions were confirmed in vitro 445 UMP-enolpyruvyltransferase NikO, S. tendae 62, 71 CAC80913 PolQ2 188 Adenylate kinase NikN, S. tendae 59, 68 CAC80912 PolQ1 419 Membrane protein NikN, S. tendae 47, 61 CAC80912 PolP 280 Acetylglutamate kinase SCO1578, S. coelicolor A3(2) 55, 68 NP_625855 PolOaFunctions were confirmed in vitro 573 Carbamoyltransferase NodU, Sinorhizobium sp. 49, 64 CAH04371 PolN 168 Amino acid N-acetyltransferase ArgA, Rhodoferax ferrireducens 30, 46 YP_523360 PolM 255 Short chain dehydrogenase PA4907, P. aeruginosa PAO1 49, 67 NP_253594 PolL 243 Unknown ORF3, Agrobacterium vitis 35, 51 ABG82019 PolK 213 Hydroxylase SanC, S. ansochromogenes 58, 69 AAF61921 PolJ 273 Phosphatase SanB, S. ansochromogenes 63, 71 AAF61216 PolI 380 Aminotransferase NikK, S. tendae 61, 69 CAC80909 PolH 469 Radical S-adenosylmethionine protein NikJ, S. tendae 70, 80 CAC80908 PolG 430 Amide synthetase NikS, Streptomyces tendae 57, 70 CAC11141 PolF 275 Molybdopterin oxidoreductase Aave_0767, Acidovorx avenae subsp. citrulli 28, 41 YP_969139 PolE 370 Unknown SAML1083, Streptomyces ambofaciens 30, 46 CAJ90069 PolD 215 Hydroxylase NikI, S. tendae 50, 65 CAC80907 PolC 204 Hydroxylase SanF, S. ansochromogenes 29, 49 AAF74976 PolY 962 Regulator ORF4, Streptomyces echinatus 39, 53 ABL09952 PolR 1111 Regulator ORFR, S. tendae 55, 66 CAC80806a Functions were confirmed in vitro Open table in a new tab Genes Essential for the Biosynthesis of the Polyoxin Nucleoside Skeleton—Six noncontiguous genes (polA, -D, -H, -I, -J, and -K) were identified as similar to an isolated gene (nikO) and five contiguous genes (nikI-nikM) involved in the biosynthesis of nikkomycin nucleoside skeleton, and were thus deduced to be responsible for the counterpart biosynthesis of the polyoxin nucleoside skeleton. PolA shows significant homology with NikO and SanX (62 and 61% identity, respectively), both independently essential for biosynthesis of nikkomycin in S. tendae Tü901 and S. ansochromogenes (9Liu G. Tian Y. Yang H. Tan H. Mol. Microbiol... 2005; 55: 1855-1866Google Scholar), likely to act as enolpyruvyltransferases (13Lauer B. Sussmuth R. Kaiser D. Jung G. Bormann C. J. Antibiot. (Tokyo).. 2000; 53: 385-392Google Scholar, 16Ginj C. Ruegger H. Amrhein N. Macheroux P. ChemBioChem.. 2005; 6: 1974-1976Google Scholar). PolD exhibits 50% identity and 65% similarity to NikI, but their precise roles in the biosynthesis of the nucleoside skeleton, either for nikkomycin or polyoxin, remain ambiguous. PolH resembles NikJ, a radical S-adenosylmethionine family protein postulated to catalyze diverse reactions, including formation of cyclized intermediate. PolI is 61% identical to NikK, and they both strongly resemble the histidinol-phosphate/aromatic aminotransferase of Magnetospirillum magneticum AMB-1. The significant resemblance of PolJ (with a protein tyrosine/serine phosphatase domain identical to NikL) and PolK (identical to NikM, a hydroxylase) strongly suggests that they should play similar roles in the biosynthesis of the nucleoside skeletons for polyoxin and/or nikkomycin. Genes for the Biosynthesis of CPOAA and POIA—Five genes (polL–P) seem to be involved in CPOAA biosynthesis, of which PolM displayed significant homology with a probable short chain dehydrogenase of Pseudomonas aeruginosa PAO1, PolN with ∼30% identity to ArgA of Rhodoferax ferrireducens, PolP with considerable homology with the corresponding protein of S. coelicolor A3(2) involved in arginine biosynthesis (30Hindle Z. Callis R. Dowden S. Rudd B.A. Baumberg S. Microbiology.. 1994; 140: 311-320Google Scholar), and PolO with significant homology with NodU, a carbamoyltransferase of Sinorhizobium sp. (31Van Rhijn P. Desair J. Vlassak K. Vanderleyden J. Appl. Environ. Microbiol... 1994; 60: 3615-3623Google Scholar). No obvious PolL homolog was found in the data base. Three genes (polC, -E, and -F) were assumed to be responsible for the biosynthesis of POIA, a distinctive moiety present in polyoxin. PolC exhibits 29% identity with SanF, a putative hydroxylase involved in nikkomycin biosynthesis in S. ansochromogenes (32Chen W. Zeng H. Tan H. Curr. Microbiol... 2000; 41: 312-316Google Scholar), whereas PolE shows no obvious similarity to proteins in the data base. Meanwhile, PolF shows only a marginal homology with Ave_0767 of Acidovorax aveenae as a putative molybdopterin oxidoreductase. Genes for the Modification of C-5 of the Nucleoside Skeleton—The methylation modification is very likely to be governed by PolB as a result of its significant homology with ThyX (a thymidylate synthase) of several bacteria, including those from S. coelicolor A3(2) (60% identity) (28Bentley S.D. Chater K.F. Cerdeno-Tarraga A.M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbinowitsch E. Rajandream M.A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature.. 2002; 417: 141-147Google Scholar) and S. avermitilis (58% identity) (29Omura S. Ikeda H. Ishikawa J. Hanamoto A. Takahashi C. Shinose M. Takahashi Y. Horikawa H. Nakazawa H. Osonoe T. Kikuchi H. Shiba T. Sakaki Y. Hattori M. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 12215-12220Google Scholar). Apart from polB, no other gene(s) related to hydroxylation/carboxylation of the methyl group of the polyoxin nucleoside skeleton was/were identified in the pol gene cluster. Genes Involved in Assembly, Regulation, and Transport—Three moieties, the nucleoside skeleton, CPOAA, and POIA, seem to be independently synthesized and assembled into polyoxin A, likely by PolG showing significant homology with NikS (SanS) (57% identity), a putative amide synthetase with confirmed ATPase activity (33Li Y. Zeng H. Tan H. Curr. Microbiol... 2004; 49: 128-132Google Scholar), as a final step. Two candidate regulatory genes, polY and polR, were identified. polY encodes a putative member of the AfsR family of the two-component response regulators, whereas polR seems to encode a pathway-specific regulator with considerable homology with SanG (OrfR in S. tendae), a positive regulator affecting both nikkomycin biosynthesis and colony devel