Title: Enhancement and Selective Production of Phoslactomycin B, a Protein Phosphatase IIa Inhibitor, through Identification and Engineering of the Corresponding Biosynthetic Gene Cluster
Abstract: Phoslactomycins (PLMs), potent and selective inhibitors of serine threonine phosphatases, are of interest for their antitumor and antiviral activity. Multiple analogs and low titers in the fermentation process have hampered the development of this class of natural products. The entire 75-kb PLM biosynthetic gene cluster of Streptomyces sp. HK-803 was cloned, sequenced, and analyzed. The loading domain and seven extension modules of the PLM polyketide synthase generate an unusual linear unsaturated polyketide chain containing both E- and Z-double bonds from a cyclohexanecarboxylic acid (CHC) primer. Hydroxylation of the CHC-derived side chain of the resulting PLM-B by PlmS 2, and a subsequent esterification, produces the remaining PLM analogs. A new PCR targeting technology allowed rapid and facile allelic replacement of plmS 2. The resulting mutant selectively produced the PLM-B, at 6-fold higher titers than the wild type strain. This mutant and the biosynthetic gene cluster will facilitate engineered microbial production of hybrid PLMs with improved properties. Phoslactomycins (PLMs), potent and selective inhibitors of serine threonine phosphatases, are of interest for their antitumor and antiviral activity. Multiple analogs and low titers in the fermentation process have hampered the development of this class of natural products. The entire 75-kb PLM biosynthetic gene cluster of Streptomyces sp. HK-803 was cloned, sequenced, and analyzed. The loading domain and seven extension modules of the PLM polyketide synthase generate an unusual linear unsaturated polyketide chain containing both E- and Z-double bonds from a cyclohexanecarboxylic acid (CHC) primer. Hydroxylation of the CHC-derived side chain of the resulting PLM-B by PlmS 2, and a subsequent esterification, produces the remaining PLM analogs. A new PCR targeting technology allowed rapid and facile allelic replacement of plmS 2. The resulting mutant selectively produced the PLM-B, at 6-fold higher titers than the wild type strain. This mutant and the biosynthetic gene cluster will facilitate engineered microbial production of hybrid PLMs with improved properties. Streptomyces produce a series of structurally novel antitumor agents that contain an unusual phosphate ester, an α,β-unsaturated δ-lactone and either a conjugated linear diene or triene chain (1Lewy D.S. Gauss C.M. Soenen D.R. Boger D.L. Curr. Med. Chem. 2002; 9: 2005-2032Crossref PubMed Scopus (137) Google Scholar). Fostriecin (Fig. 1) and the structurally related PD 113,270 and PD 113,271 are three related natural products produced by Streptomyces pulveraceus subsp. fostreus ATCC 31906 (2Tunac J.B. Graham B.D. Dobson W.E. J. Antibiot. (Tokyo). 1983; 36: 1595-1600Crossref PubMed Scopus (103) Google Scholar, 3Stampwala S.S. Bunge R.H. Hurley T.R. Willmer N.E. Brankiewicz A.J. Steinman C.E. Smitka T.A. French J.C. J. Antibiot. (Tokyo). 1983; 36: 1601-1605Crossref PubMed Scopus (126) Google Scholar). Fostriecin inhibits DNA topoisomerase II (IC50, l40 mm) (4de Jong R.S. Mulder N.H. Uges D.R. Sleijfer D.T. Hoppener F.J. Groen H.J. Willemse P.H. van der Graaf W.T. de Vries E.G. Br. J. Cancer. 1999; 79: 882-887Crossref PubMed Scopus (56) Google Scholar) and is also a potent and selective inhibitor of protein phosphatases 1 (PP1), 1The abbreviations used are: PP, protein phosphatases; PLM, phoslactomycin; PKS, polyketide synthase; CHC, cyclohexanecarboxylic acid; ACP, acyl carrier protein; TE, thioesterase; ORF, open reading frame.1The abbreviations used are: PP, protein phosphatases; PLM, phoslactomycin; PKS, polyketide synthase; CHC, cyclohexanecarboxylic acid; ACP, acyl carrier protein; TE, thioesterase; ORF, open reading frame. PP2A, and PP4 (IC50, 45 mm, 1.5 nm, and 3.0 nm, respectively) (1Lewy D.S. Gauss C.M. Soenen D.R. Boger D.L. Curr. Med. Chem. 2002; 9: 2005-2032Crossref PubMed Scopus (137) Google Scholar, 5Boger D.L. Ichikawa S. Zhong W. J. Am. Chem. Soc. 2001; 123: 4161-4167Crossref PubMed Scopus (119) Google Scholar). The latter activity is attributed to the efficacious in vivo antitumor activity of fostriecin, as well as its in vitro activity against leukemia, lung, breast, and ovarian cancer. The same activity likely also permits fostriecin to limit myocardial infarct size and to protect cardiomyocytes during ischemia (6Armstrong S.C. Gao W. Lane J.R. Ganote C.E. J. Mol. Cell Cardiol. 1998; 30: 61-73Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 7Weinbrenner C. Baines C.P. Liu G.S. Armstrong S.C. Ganote C.E. Walsh A.H. Honkanen R.E. Cohen M.V. Downey J.M. Circulation. 1998; 98: 899-905Crossref PubMed Scopus (74) Google Scholar, 8Armstrong S.C. Kao R. Gao W. Shivell L.C. Downey J.M. Honkanen R.E. Ganote C.E. J. Mol. Cell Cardiol. 1997; 29: 3009-3024Abstract Full Text PDF PubMed Scopus (40) Google Scholar). Recent evidence suggests that PP2A has a role in regulating cell death by apoptosis, in the activation of natural killer cells and cytotoxic T-lymphocytes involved in tumor surveillance, and even in the transcription and replication of human immunodeficiency virus-1 (HIV1) (9Faulkner N.E. Lane B.R. Bock P.J. Markovitz D.M. J. Virol. 2003; 77: 2276-2281Crossref PubMed Scopus (25) Google Scholar). For these reasons selective protein phosphatase inhibition has been suggested as a clinically unexplored novel mechanism worthy of pursuit for the introduction of a new class of antitumor or even antiviral agents (1Lewy D.S. Gauss C.M. Soenen D.R. Boger D.L. Curr. Med. Chem. 2002; 9: 2005-2032Crossref PubMed Scopus (137) Google Scholar, 5Boger D.L. Ichikawa S. Zhong W. J. Am. Chem. Soc. 2001; 123: 4161-4167Crossref PubMed Scopus (119) Google Scholar, 9Faulkner N.E. Lane B.R. Bock P.J. Markovitz D.M. J. Virol. 2003; 77: 2276-2281Crossref PubMed Scopus (25) Google Scholar).Phoslactomycins (PLMs) (also called phosphazomycins or phospholines) and leustroducsins (Fig. 1) are structurally related natural products. The principal differences from fostriecin are replacement of the C-8 methyl substituent by ethylamine and the terminal allylic alcohol by a cyclohexane ring. Like fostriecin, these compounds are produced in multiple forms (Fig. 1). PLM-B (phospholine) contains a fully reduced cyclohexyl ring, whereas the other PLMs and the leustroducsins contain a hydroxyl substituent (C-18) esterified with a wide array of carboxylic acids (ranging in length from 4 to 9 carbons) (10Fushimi S. Furihata K. Seto H. J. Antibiot. (Tokyo). 1989; 42: 1026-1036Crossref PubMed Scopus (76) Google Scholar, 11Kohama T. Enokita R. Okazaki T. Miyaoka H. Torikata A. Inukai M. Kaneko I. Kagasaki T. Sakaida Y. Satoh A. et al.J. Antibiot. (Tokyo). 1993; 46: 1503-1511Crossref PubMed Scopus (67) Google Scholar, 12Kohama T. Nakamura T. Kinoshita T. Kaneko I. Shiraishi A. J. Antibiot. (Tokyo). 1993; 46: 1512-1519Crossref PubMed Scopus (66) Google Scholar, 13Tomiya T. Uramoto M. Isono K. J. Antibiot. (Tokyo). 1990; 43: 118-121Crossref PubMed Scopus (31) Google Scholar). The phoslactomycins and leustroducsins all exhibit antifungal activity (1Lewy D.S. Gauss C.M. Soenen D.R. Boger D.L. Curr. Med. Chem. 2002; 9: 2005-2032Crossref PubMed Scopus (137) Google Scholar, 10Fushimi S. Furihata K. Seto H. J. Antibiot. (Tokyo). 1989; 42: 1026-1036Crossref PubMed Scopus (76) Google Scholar, 13Tomiya T. Uramoto M. Isono K. J. Antibiot. (Tokyo). 1990; 43: 118-121Crossref PubMed Scopus (31) Google Scholar, 14Uramoto M. Shen Y.C. Takizawa N. Kusakabe H. Isono K. J. Antibiot. (Tokyo). 1985; 38: 665-668Crossref PubMed Scopus (22) Google Scholar). More importantly, these compounds like fostriecin are potent and highly selective inhibitors of PP2A (IC50 values raging from 3.7 to 5.8 μm) as compared with PP1 (IC50 > 1 mm) (15Usui T. Marriott G. Inagaki M. Swarup G. Osada H. J. Biochem. 1999; 125: 960-965Crossref PubMed Scopus (52) Google Scholar). Most recently, the PP2A inhibition activity of phoslactomycin has been shown to inhibit tumor metastasis through augmentation of natural killer cells (16Kawada M. Kawatsu M. Masuda T. Ohba S. Amemiya M. Kohama T. Ishizuka M. Takeuchi T. Int. Immunopharmacol. 2003; 3: 179-188Crossref PubMed Scopus (41) Google Scholar).The unique and selective biological activity of this class of natural products has attracted considerable interest in recent years. Phase I clinical trials of fostriecin were suspended before either dose-limiting toxicities or therapeutic plasma levels were attained because of inherent drug instability and unpredictable purity in the clinical supply of the natural product (1Lewy D.S. Gauss C.M. Soenen D.R. Boger D.L. Curr. Med. Chem. 2002; 9: 2005-2032Crossref PubMed Scopus (137) Google Scholar, 4de Jong R.S. Mulder N.H. Uges D.R. Sleijfer D.T. Hoppener F.J. Groen H.J. Willemse P.H. van der Graaf W.T. de Vries E.G. Br. J. Cancer. 1999; 79: 882-887Crossref PubMed Scopus (56) Google Scholar). In an attempt to address these limitations and further develop this class of novel antitumor agents, no less than six elegant total syntheses of fostriecin have been developed over a short 2-year period (5Boger D.L. Ichikawa S. Zhong W. J. Am. Chem. Soc. 2001; 123: 4161-4167Crossref PubMed Scopus (119) Google Scholar, 17Fujii K. Maki K. Kanai M. Shibasaki M. Org. Lett. 2003; 5: 733-736Crossref PubMed Scopus (117) Google Scholar, 18Esumi T. Okamoto N. Hatakeyama S. Chem. Commun. (Camb.). 2002; : 3042-3043Crossref PubMed Scopus (67) Google Scholar, 19Wang Y.G. Kobayashi Y. Org. Lett. 2002; 4: 4615-4618Crossref PubMed Scopus (61) Google Scholar, 20Reddy Y.K. Falck J.R. Org. Lett. 2002; 4: 969-971Crossref PubMed Scopus (86) Google Scholar, 21Miyashita K. Ikejiri M. Kawasaki H. Maemura S. Imanishi T. Chem. Commun. (Camb.). 2002; : 742-743Crossref PubMed Scopus (53) Google Scholar). A complementary approach for developing these agents can come through gene manipulation of the natural biosynthetic process. In all cases, the production of numerous analogs complicates the isolation process and the purity of the product, as well as negatively impacting yields (2Tunac J.B. Graham B.D. Dobson W.E. J. Antibiot. (Tokyo). 1983; 36: 1595-1600Crossref PubMed Scopus (103) Google Scholar, 14Uramoto M. Shen Y.C. Takizawa N. Kusakabe H. Isono K. J. Antibiot. (Tokyo). 1985; 38: 665-668Crossref PubMed Scopus (22) Google Scholar, 22Fushimi S. Nishikawa S. Shimazu A. Seto H. J. Antibiot. (Tokyo). 1989; 42: 1019-1025Crossref PubMed Scopus (92) Google Scholar). These analogs are based on variations in the level of hydroxylation and, in the case of the PLMs and leustroducsins, esterification of the resulting secondary alcohol with a series of different carboxylic acids (this modification does not significantly impact either antifungal activity or binding to PP2A). Using a PLM-producing strain, Streptomyces sp. HK-803, we have carried out an incorporation study of [7-13C]CHC, which suggests that PLM-A and PLM C–F are derived from a hydroxylation of PLM-B. The entire 75 kb of the phoslactomycin biosynthetic gene cluster has been identified and sequenced, including a gene, plmS 2, encoding the cyctochrome P-450 monooxygenase responsible for this hydroxylation. The application of a new PCR targeting technology (23Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar) in this strain allowed rapid gene replacement of plmS 2. The resulting mutant strain, NP1, produces only PLM-B, at 6-fold higher levels than the wild type strain. This mutant will serve as a starting point for engineering strains capable of producing fostriecin and PLM analogs.EXPERIMENTAL PROCEDURESStrains and Culture Conditions—All Escherichia coli strains used in this study were grown following standard protocols (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The PLM producer Streptomyces sp. HK-803 (13Tomiya T. Uramoto M. Isono K. J. Antibiot. (Tokyo). 1990; 43: 118-121Crossref PubMed Scopus (31) Google Scholar) was grown at 28 °C in SY medium (1.0% soluble starch, 0.1% yeast extract, 0.1% N-Z-amine, type A) at pH 7.0. The PLM production medium was 2.0% glucose, 0.1% beef extract, 1.0% soybean flour, 0.2% NaCl, 0.005% K2HPO4, and 0.2% l-phenylalanine at pH 7.0. A loop-full of Streptomyces sp. HK-803 spores (from an SY agar plate) was inoculated into a baffled 500-ml flask containing 70 ml of the production medium and was incubated at 28 °C in the dark for 96 h on a rotary shaker at 170 rpm.Analyses of PLM Production—The culture filtrates and mycelial extracts of Streptomyces sp. HK-803 wild type and the mutant NP1 were worked up as described previously (13Tomiya T. Uramoto M. Isono K. J. Antibiot. (Tokyo). 1990; 43: 118-121Crossref PubMed Scopus (31) Google Scholar). HPLC was performed on a system equipped with a Waters™ 600 pump and a Waters 996 photodiode array detector under the following conditions: column, Senshu Pak PEGASIL ODS (4.6 × 250 mm for analytical HPLC and 20 × 250 mm for preparative HPLC); solvent, acetonitrile, 0.05% HCO2H (40/60); flow rate, 1 ml/min for analytical HPLC and 9 ml/min for preparative HPLC; detection at UV 235 nm. Pure PLM standards were used to generate a standard curve for determining PLM production in both the wild type and NP1 mutant. The PLMs were also identified by co-injections with the corresponding standards and by LC-MS analyses. LC-MS spectra (positive turbo-ion spray ionization mode; HPLC, Hewlett-Packard Series 1100; column, 150 × 2.1 mm RP 183.5-mm column from Waters; mobile phase, 40:60 CH3CN-water (containing 0.05% HCO2H) at a flow rate 0.2 ml/min) were taken on a PerkinElmer Life Sciences SCIEX API 2000 pneumatically assisted electrospray triple quadrupole mass spectrometer.Incorporation of [7-13C]CHC into PLMs—[7-13C]CHC was synthesized from cyclohexanone and K13CN via cyclohexanecarbonitrile as described previously (25Jiricny J. Orere D.M. Reese C.B. J. Chem. Soc. Perkin Trans. I. 1980; : 1487-1492Crossref Google Scholar, 26Orere D.M. Reese C.B. J. Chem. Soc. Chem. Commun. 1977; : 280-281Crossref Google Scholar): 1H NMR (270 MHz, CDCl3) δ 1.20–2.00 (10H, m), 2.33 (1H, tdt, J H-H = 15.1, 2 J C-H = 7.1, J H-H = 3.6 Hz), 11.4 (1H, bs); 13C-NMR (67.5 MHz, CDCl3) δ 25.4, (d, 3 J C-C = 3.85 Hz, C-3), 25.8, 28.8 (d, 2 J C-C = 1.08 Hz, C-2), 43.0 (1 J C-C = 55.0 Hz, C-1), 182.6 (enhanced signal, C-7).The labeled CHC (10 mg) was dissolved in EtOH (1 ml) and added directly to each of 10 flasks containing 70 ml of production medium at 30 h after inoculation. LC-MS analyses (see “Experimental Procedures”) of the resulting PLMs allowed an estimation of the level of incorporation of [7-13C]CHC. For each PLM the ratio of the [M+1] and [M+2] relative to [M], the abundance of the molecular ion (m/z), was determined. A similar analysis was carried out with unlabeled PLMs and allowed the contributions of naturally abundant 13C and [7-13C]CHC to be separated in the standard manner. The amount of each 13C-labeled PLM derived from [7-13C]CHC was determined as a percentage of that entire analog pool. The location of the 13C label was confirmed by 13C NMR analyses of HPLC purified PLM-B, -E, and -F (see “Experimental Procedures”) using previously published 13C assignments (10Fushimi S. Furihata K. Seto H. J. Antibiot. (Tokyo). 1989; 42: 1026-1036Crossref PubMed Scopus (76) Google Scholar).Cloning and Sequencing of the PLM Biosynthetic Gene Cluster—The total genomic DNA of Streptomyces sp. HK-803 (grown on SY medium) was prepared according to the cetyltrimethylammonium bromide procedure for the isolation of genomic DNA in the manual of Practical Streptomyces genetics (27Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, United Kingdom2000: 170-171Google Scholar). A genomic library was constructed by using Supercos-1 cosmid vector as recommended in the manufacturer's protocol (Stratagene). About 2000 cosmid clones were probed by using the DNA of a digoxigenin-labeled chcA gene (28Wang P. Denoya C.D. Morgenstern M.R. Skinner D.D. Wallace K.K. DiGate R. Patton S. Banavali N. Schuler G. Speedie M.K. Reynolds K.A. J. Bacteriol. 1996; 178: 6873-6881Crossref PubMed Google Scholar). Preparation of the digoxigenin probes and the subsequent hybridization and detection were performed as recommended in the manufacturer's protocol (Roche Applied Science). The overlapping cosmid clones identified by the chcA probe were sequenced to completion using the TOPO shotgun subcloning kit (Invitrogen). Automated DNA sequencing was performed on an ABI Prism 3700 DNA sequencer at the DNA core facility of the Medical College of Virginia, Virginia Commonwealth University. The DNA sequences were assembled using SeqMan II (DNAStar Inc.). The assembled DNA and deduced protein sequences were analyzed with MacVector 7.0 software (Accelrys) and FramePlot (29Ishikawa Hotta K. FEMS Microbiol. Lett. 1999; 174: 251-253Crossref PubMed Google Scholar) and were compared with sequences in the public data bases using the BLAST suite of programs (30Altschul S.F. Gish W. Miller W. Meyers W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69138) Google Scholar).Targeted Disruption of plmS2—The plmS 2 gene of the PLM biosynthetic gene cluster was disrupted by using the recently developed PCR-targeted Streptomyces gene replacement method (23Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar). The aac (3)IV resistance marker and oriT were amplified from the pIJ 773 disruption cassette (23Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar) using the primers PLMS 2 -F 5′-CCGCCGCCCCGAGCACGA-AGAGACGGCGTCCTCCGCATGattccggggatccgtcgacc-3′ and PLMS 2 -R 5′-CCAGG-ACGACCGGGCCGGTTCAGCGTGCGGGAACGCTCAtgtaggctggagctgcttc-3′ (pIJ773 homologous sequence is in lowercase letters). The resulting PCR product was used to replace plmS 2 first in cosmid clone 3A11 and then in Streptomyces sp. HK-803 following the established methodologies (23Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1191) Google Scholar), replacing mannitol soy agar with SY agar. Allelic replacement of the plmS 2 gene in the NP1 mutant was confirmed by Southern hybridization analysis, and PCR amplification and sequencing.RESULTS AND DISCUSSIONIncorporation Studies Establish CHC as a Precursor to all PLMs—Analysis of the PLM-B structure suggested that it is likely assembled by a type I modular polyketide synthase (PKS) using a CHC starter unit. The other PLMs and the leustroducsins may be derived by hydroxylation of this PLM-B or by use of a cis-3-hydroxy-CHC starter unit. A CHC-derived moiety has previously been observed as a side chain in the antifungal agent ansatrienin and shown to be derived from shikimic acid (31Moore B.S. Cho H. Casati R. Kennedy E. Reynolds K.A. Mocek U. Beale J.M. Floss H.G. J. Am. Chem. Soc. 1993; 115: 5254-5266Crossref Scopus (65) Google Scholar, 32Casati R. Beale J.M. Floss H.G. J. Am. Chem. Soc. 1987; 109: 8102-8104Crossref Scopus (27) Google Scholar). LC-MS analysis of the PLM mixture obtained from the feeding of [7-13C]CHC to produce cultures of Streptomyces sp. HK-803 showed that this compound was efficiently converted into all PLMs. More than 50% of the entire pool of PLM-E, which contains two cyclohexyl moieties, was labeled by at least one molecule of [7-13C]CHC. Approximately half of this level of labeling (21–28%) was observed for PLM-A, PLM-D, PLM-E, and PLM-F, consistent with these analogs containing only one putative CHC-derived unit. Curiously, almost 48% of the PLM-B was labeled by [7-13C]CHC. Consistent with these observations, a 13C NMR spectra (125 MHz, CD3OD) of the purified PLM-E showed strongly enhanced signals at the predicted sites, C-15 (δ 139.9 ppm) and C-1′ (δ 177.3 ppm), indicating that CHC is the precursor of both cyclohexane moieties. Strong and exclusive enrichments of the C-15 (δ 138.2 ppm) was observed in 13C NMR analyses of purified PLM-B and PLM-F. The level of enrichment in all cases was comparable with that determined from LC-MS analyses. These observations demonstrate that a coenzyme A-activated CHC (CHC-CoA) is the likely starter unit for the biosynthesis of all PLMs. A pathway from shikimic acid to CHC-CoA has been delineated in the ansatrienin producer, Streptomyces collinus (31Moore B.S. Cho H. Casati R. Kennedy E. Reynolds K.A. Mocek U. Beale J.M. Floss H.G. J. Am. Chem. Soc. 1993; 115: 5254-5266Crossref Scopus (65) Google Scholar). The corresponding biosynthetic genes have recently been identified (33Cropp T.A. Wilson D.J. Reynolds K.A. Nat. Biotechnol. 2000; 18: 980-983Crossref PubMed Scopus (80) Google Scholar, 34Chen S. von Bamberg D. Hale V. Breuer M. Hardt B. Muller R. Floss H.G. Reynolds K.A. Leistner E. Eur. J. Biochem. 1999; 261: 98-107Crossref PubMed Scopus (79) Google Scholar).Isolation and Characterization of the PLM Biosynthetic Gene Cluster—The PLM biosynthetic gene cluster from the Streptomyces sp. HK-803 was identified from a cosmid library of genomic DNA using the chcA gene (28Wang P. Denoya C.D. Morgenstern M.R. Skinner D.D. Wallace K.K. DiGate R. Patton S. Banavali N. Schuler G. Speedie M.K. Reynolds K.A. J. Bacteriol. 1996; 178: 6873-6881Crossref PubMed Google Scholar) from CHC-CoA biosynthetic gene cluster of S. collinus as a probe. A cosmid clone, 3A11, thus identified was used as a probe to identify two overlapping cosmids, 10B4 and 3E5 (Fig. 2). These three cosmids spanning 97 kb of the Streptomyces sp. HK-803 genome were sequenced and analyzed. This sequence analysis revealed a 75-kb region containing 29 open reading frames (ORFs) that appear to be associated with PLM biosynthesis. The flanking region contained a series of ORFs that are highly homologous (>80% identity at the amino acid sequence level) with and have an organization similar to the primary metabolic genes identified through sequencing of the S. coelicolor genome (35Bentley 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: 141-147Crossref PubMed Scopus (2555) Google Scholar), which aided in delimiting the PLM biosynthetic gene cluster. The sequence analysis revealed the existence of the complete set of highly conserved CHC-CoA biosynthetic genes (plmJK, plmL, chcA, plmM) (28Wang P. Denoya C.D. Morgenstern M.R. Skinner D.D. Wallace K.K. DiGate R. Patton S. Banavali N. Schuler G. Speedie M.K. Reynolds K.A. J. Bacteriol. 1996; 178: 6873-6881Crossref PubMed Google Scholar, 33Cropp T.A. Wilson D.J. Reynolds K.A. Nat. Biotechnol. 2000; 18: 980-983Crossref PubMed Scopus (80) Google Scholar) as well as plmI (encoding a 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) responsible for formation of the CHC-CoA starter unit. As in S. collinus, the chcB gene encoding the Δ2,3-enoyl CoA isomerase responsible for catalyzing the penultimate step in the CHC-CoA pathway does not appear to be located close to the other CHC-CoA biosynthetic genes or within the antibiotic biosynthetic gene cluster (36Patton S.M. Cropp T.A. Reynolds K.A. Biochemistry. 2000; 39: 7595-7604Crossref PubMed Scopus (21) Google Scholar). A total of six large ORFs encoding the core PLM PKS were also identified (Fig. 3). PLM1 contains an initiation domain, presumably responsible for loading the CHC-CoA, and the first extension module (Fig. 3), whereas PLM2–3 has two modules containing the appropriate predicted catalytic activitities for catalyzing the second and third extension steps. Each of the remaining components, PLM4–PLM7, contains a single module. PLM7 has a thioesterase domain, consistent with a role in catalyzing the last extension step and subsequent release of the polyketide chain by formation of the α,β-unsaturated δ-lactone (formation of a six-member lactone is unusual, as most thioesterase domains studied to date catalyze formation of large 12–14 macrolactone structures under natural conditions) (37Tsai S.C. Lu H. Cane D.E. Khosla C. Stroud R.M. Biochemistry. 2002; 41: 12598-12606Crossref PubMed Scopus (98) Google Scholar, 38Lu H. Tsai S.C. Khosla C. Cane D.E. Biochemistry. 2002; 41: 12590-12597Crossref PubMed Scopus (60) Google Scholar). Two of the PKS polypeptides PLM4 and PLM6, contain the same catalytic domains, which precludes unambiguous assignment in this process. One of many interesting features of the PLM PKS cluster is that it contains only three dehydratase domains, and yet it produces a compound with three Z- and one E-double bonds (PLM7 does not contain the predicted dehydratase domain). Virtually all known cases in which a PKS modules has a ketoreductase and a dehydratase activity result in the formation of E-double bonds (39Reid R. Piagentini M. Rodriguez E. Ashley G. Viswanathan N. Carney J. Santi D.V. Hutchinson C.R. McDaniel R. Biochemistry. 2003; 42: 72-79Crossref PubMed Scopus (259) Google Scholar). It has been suggested that a stereochemical configuration of the product derived from the ketoreductase domain may control the double bond geometry. A diagnostic Asp residue has been proposed for predicting the stereospecificity of PKS ketoreductase domains and, by extension, the double bond configuration for products from cognate dehydratase domains (39Reid R. Piagentini M. Rodriguez E. Ashley G. Viswanathan N. Carney J. Santi D.V. Hutchinson C.R. McDaniel R. Biochemistry. 2003; 42: 72-79Crossref PubMed Scopus (259) Google Scholar). We constructed a multiple alignment of the seven ketoreductase domains of the PLM PKS and found that this model alone was only partially predictive of the stereochemical configuration of the PLM product.Fig. 2The phoslactomycin biosynthetic gene cluster. The genes plm1–plm8 encode the polyketide synthase (see Fig. 3 for more details). Genes proposed to be involved in providing the precursors CHC-CoA and butyryl CoA (plmT 7) are shown in light blue. Regulatory genes (dark blue) and an ABC transporter (green) are shown. Genes depicted in yellow encode enzymes proposed to be involved in postpolyketide modification and include two cytochrome P-450 monooxygenases (plmS 2, plmT 4), an oxidoreductase (plmT 8), an aminotransferase (plmT 1), an a kinase (plmT5). The plmS 2 mutant (strain NP1) produces only PLM-B (see Fig. 4). The sequence of the entire gene cluster and the proposed function for individual ORFs is summarized in GenBank™ under accession number AY354515.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Proposed role for the six polypetides of the PLM PKS in PLM-B biosynthesis. The structures of the PKS-bound intermediates and the product released from the PKS (in parentheses) are hypothetical.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The PLM gene cluster also contains genes that encode a separate type II TE protein (plmT 8), recently established to have an important role in the editing and proofreading of PKSs (40Kim B.S. Cropp T.A. Beck B.J. Sherman D.H. Reynolds K.A. J. Biol. Chem. 2002; 277: 48028-48034Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and a crotonyl-CoA reductase (plmT 7), presumably required for providing the ethylmalonyl-CoA extender unit used in the fourth and sixth extension steps. The plmT 5 gene product has some sequence identity with homoserine kinases and is likely responsible for phosphorylation of the C-9 hydroxyl group and for providing biological activity to PLM. Analysis of the gene cluster also predicts an oxidoreductase (plmT 8) and aminotransferase (plmT 1), likely involved in the replacement of a hydroxyl group of a C-8 hydroxyethyl side chain with an amine. The gene cluster also contains numerous other genes involved in regulation and resistance. A detailed analysis of the entire PLM gene cluster will be described elsewhere.PCR-targeted Gene Disruption of plmS2—Analysis of the PLM biosynthetic gene cluster, together with the results of the biosynthetic incorporation study, indicated that three hydroxylation steps (C-18, C-8, and the C-8 ethyl side chain) occur after assembly of the polyketide chain from the CHC-derived starter unit. Analysis of the PLM biosynthetic gene cluster revealed two ORFs, plmT 4 and plmS 2, that both encode proteins with high sequence similarity to each other (38% identity and 53% similarity) as well as cytochrome P-450 monooxygenases from several microorganisms, including those involved in the modification of the natural products of streptomycetes (41Xue Y. Wilson D. Zhao L. Liu H. Sherman D.H. Chem. Biol. 1998; 5: 661-667Abstract Full Text PDF PubMed Scopus (112) Google Scholar). We reasoned that replacement of one of these genes would provide a strain blocked in hydroxylation of C-18 of PLM. With the caveat that the other hydroxylations are catalyzed by the second monooxygenase, the mutant strain should produce PLM-B exclusively.There are numerous different app