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• W2017109849 abstract "Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to β-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids. Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to β-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids. Type III PKSs 2The abbreviations used are: PKS, polyketide synthase; HPLC, high performance liquid chromatography; LC-APCIMS, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry; MS, mass spectrometry. 2The abbreviations used are: PKS, polyketide synthase; HPLC, high performance liquid chromatography; LC-APCIMS, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry; MS, mass spectrometry. are structurally and functionally simple PKSs that catalyze the synthesis of aromatic polyketides in both plants and microorganisms (1Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (712) Google Scholar). We previously found that a type III PKS, RppA, in the Gram-positive, filamentous bacterium Streptomyces griseus catalyzes the synthesis of 1,3,6,8-tetrahydroxynaphthalene by using malonyl-CoA as a starter, carrying out four successive extensions with malonyl-CoA and cyclizing the resulting pentaketide to the naphthalene scaffold (2Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (234) Google Scholar). This was the first report of a functional type III PKS from bacteria. Since then, genome projects of bacteria have predicted that type III PKSs are distributed widely not only in Streptomyces but also other various bacteria. For example, ArsB and ArsC, both of which are type III PKSs in Azotobacter vinelandii, catalyze the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the cyst membrane (3Funa N. Ozawa H. Hirata A. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6356-6361Crossref PubMed Scopus (119) Google Scholar).We have recently completed the genome project for S. griseus 3Ohnishi, Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita, A., Hattori, M., and Horinouchi, S. (2008) J. Bacteriol., in press. 3Ohnishi, Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita, A., Hattori, M., and Horinouchi, S. (2008) J. Bacteriol., in press. and found another gene encoding a type III PKS, in addition to RppA. This type III PKS was named SrsA (Streptomyces resorcinol synthesis) because of its ability to synthesize alkylresorcinols in a unique manner (see below). SrsA, showing 31% amino acid sequence identity to RppA, appeared to be a member of the proteins whose synthesis was directed by an srs operon consisting of three genes, srsA, srsB, and srsC (see Fig. 1). In this paper, we report the function of the srs operon by elucidating the in vivo and in vitro enzymatic properties of the Srs products. SrsA was responsible for synthesis of the amphiphilic skeletons of phenolic lipids, alkylresorcinols and alkylpyrones, from acyl-CoAs of various chain lengths, as determined by in vitro experiments. Consistent with this idea, overexpression of srsA in a heterologous host, Streptomyces lividans, led to the accumulation of phenolic lipids. In contrast with ArsB and ArsC in A. vinelandii, SrsA used both methylmalonyl-CoA and malonyl-CoA as extender substrates, and the rank order of assembly of the extender substrates was strictly controlled. When srsA and srsB were co-expressed in S. lividans, alkylresorcinol methyl ethers were produced, whereas co-expression of srsA, srsB, and srsC led to production of hydroxylated alkylresorcinols. These observations showed that SrsB was a methyl transferase acting on alkylresorcinols, and SrsC was a hydroxylase acting on alkylresorcinol methyl ethers.In addition to the function of the srsABC operon, we observed a possible role for the metabolites, alkylresorcinols and alkylpyrones. An S. griseus mutant deficient in the phenolic lipids synthesis was highly sensitive to penicillin G and cephalexin, inhibitors of cell wall synthesis, which suggests that the phenolic lipids, presumably integrated and orientated in the cytoplasmic membrane, confer rigidity of the membrane, thus permitting S. griseus to grow in the presence of a higher concentration of the β-lactam antibiotics. Together with the fact that a pair of genes encoding SrsA- and SrsB-like proteins is distributed widely in both Gram-positive and -negative bacteria, we speculate that the phenolic lipids play a significant, but so far unrecognized, role in the biological membranes. An attractive example is the phenolic lipids that are essential for cyst formation in A. vinelandii (3Funa N. Ozawa H. Hirata A. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6356-6361Crossref PubMed Scopus (119) Google Scholar).EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Media—Escherichia coli JM109, plasmid pUC19, restriction enzymes, and other DNA-modifying enzymes used for DNA manipulation were purchased from Takara Biochemicals (Shiga, Japan). The media, growth conditions, and general recombinant DNA techniques were described by Sambrook and co-workers (4Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). S. griseus IFO13350 was obtained from the Institute of Fermentation, Osaka (IFO). S. lividans TK21 was obtained from D. A. Hopwood (5Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). For expression of srsA, srsB, and srsC in Streptomyces, pIJ6021 containing a thiostrepton-inducible tipA promoter (6Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (127) Google Scholar) was used. pKUM10 (7Yamazaki H. Takano Y. Ohnishi Y. Horinouchi S. Mol. Microbiol. 2003; 50: 1173-1187Crossref PubMed Scopus (44) Google Scholar) with its copy number of one to two/chromosome was used as a Streptomyces vector. For production of His-tagged SrsA in S. lividans TK21, pSH19 containing an ϵ-caprolactam-inducible nitA promoter (8Herai S. Hashimoto Y. Higashibata H. Maseda H. Ikeda H. Omura S. Kobayashi M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14031-14035Crossref PubMed Scopus (104) Google Scholar) was used. S. lividans and S. griseus were cultured in yeast extract-malt extract medium (5Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar) and YMPD medium (0.2% yeast extract (Difco), 0.2% meat extract (Kyokuto), 0.4% Bacto peptone (Difco), 0.5% NaCl, 0.2% MgSO4·7H2O, 1% glucose, pH 7.2), respectively. Nucleotide sequences were determined with the Amersham Biosciences Thermo Sequenase fluorescence-labeled primer cycle sequencing kit on an automated DNA sequencer. All PCRs were conducted by using the chromosomal DNA of S. griseus IFO13350 as the template if the template is not indicated. The absence of undesired alterations during PCR was checked by nucleotide sequencing.Construction of pIJ6021-srsA (pFM1)—For construction of pFM1 containing srsA under the control of the tipA promoter in pIJ6021 (see Fig. 1), an NdeI site was introduced at the start codon of srsA by PCR with primer I: 5′-GCGGAATTCCATATGACCCGCATCGCCGCGGT-3′ (with an EcoRI site shown by underlining and the nucleotides to be changed shown by the italic letters) and primer II: 5′-GCGAAGCTTGGATCCGAGGTCATGGGTCTCTCCTTC-3′ (with a HindIII site shown by underlining, a BamHI site shown by the italic letters, and the start codon of srsB shown by the boldface letters). The 1.1-kb amplified fragment was cloned between the EcoRI and HindIII sites of pUC19, resulting in pUC19-srsA. The NdeI-BamHI fragment excised from pUC19-srsA was cloned between the NdeI and BamHI sites of pIJ6021, resulting in pFM1.Construction of pIJ6021-srsAB (pFM2)—For construction of pFM2 containing srsA and srsB (see Fig. 1), the 1.6-kb fragment was amplified by PCR with primer I and primer III (5′-GCGAAGCTTCTCGAGGCCGACGACCAGGACGTCGA-3′; with a HindIII site shown by the italic letters). The amplified fragment was cloned between the EcoRI and HindIII sites of pUC19, resulting in pUC19-srsAB. The NdeI-HindIII fragment excised from pUC19-srsAB was cloned between the NdeI and HindIII sites of pIJ6021, resulting in pFM2.Construction of pIJ6021-srsABC (pFM3)—For construction of pFM3 containing srsA, srsB, and srsC (see Fig. 1), the srsC sequence was attached to the srsAB sequence in pFM2. The 1.1-kb srsC sequence from a BamHI site located 50 bp upstream from the stop codon of srsB to the nucleotide 68 bp downstream from the stop codon of srsC was amplified by PCR with primer IV (5′-GCGGGATCCGCTGCGAGGACGG-3′; with a BamHI site shown by the italic letters) and primer V (5′-GCGAAGCTTCGACCGACGTTATGCCGGAACC-3′; with a HindIII site shown by the italic letters). The BamHI-HindIII fragment was cloned between the BamHI and HindIII sites of pUC19, resulting in pUC19-srsC. The 1.1-kb BamHI-HindIII fragment excised from pUC19-srsC was cloned between the BamHI and HindIII sites of pUC19-srsAB, resulting in pUC19-srsABC. The 2.7-kb NdeI-HindIII fragment excised from pUC19-srsABC was cloned between the NdeI and HindIII sites of pIJ6021, resulting in pFM3.Construction of pKUM10-srsA—A 0.8-kb DNA fragment from the nucleotide 480 bp upstream of the srsA start codon to a SalI site located 380 bp downstream of the srsA start codon was amplified by PCR with primer VI (5′-GCGAAGCTTGACCCCGCAGGCCAGCGAGAGC-3′; with a HindIII site shown by the italic letters) and primer VII (5′-GCGGTCGACGGAGGGGGCGGCGATG-3′; with a SalI site shown by the italic letters). The HindIII-SalI fragment was cloned between the HindIII and SalI sites of pUC19, resulting in pUC19-srsAN. The SalI-BamHI fragment excised from pUC19-srsAB was cloned between the SalI and BamHI sites of pUC19-srsAN, resulting in pUC19-srsABN. The EcoRI-BamHI fragment excised from pUC19-srsABN was cloned between the EcoRI and BamHI sites of pKUM10, resulting in pKUM10-srsA.Construction of pSH19-srsA—For adding a histidine tag to the C terminus of SrsA, srsA was cloned in pET26b by introducing an NdeI and BamHI sites in the nucleotide sequences covering the start and stop codons, respectively, by PCR. PCR was performed with primer I and primer VIII (5′-GCGAAGCTTGGATCCCAGCGCAGCAGCACCAGTTCG-3′; with a HindIII and BamHI sites shown by underlining and the italic letters, respectively). The amplified fragment was cloned between the EcoRI and HindIII sites of pUC19, resulting in pUC19-srsA26. The NdeI-BamHI fragment excised from pUC19-srsA26 was cloned between the NdeI-BamHI sites of pET26b, resulting in pET26b-srsA. The nucleotide sequence of srsA together with the histidine tag sequence in pET26b-srsA was amplified by PCR with primer IX (5′-GCGCTGCAGAGCAACGGAGGTACGGACATGACCCGCATCGCCGCGGT-3′; with a PstI site shown by the italic letters; the sequence in the boldface letters is the nitA recognition sequence, including the Shine-Dalgarno-sequence shown by underlining) (8Herai S. Hashimoto Y. Higashibata H. Maseda H. Ikeda H. Omura S. Kobayashi M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14031-14035Crossref PubMed Scopus (104) Google Scholar) and primer X (5′-GCGGGTACCTTTCGGGCTTTGTTAGCAGCCG-3′; with a KpnI site shown by the italic letters). The amplified fragment was cloned between the PstI and KpnI sites of pUC19, resulting in pUC19-srsAH. The PstI-KpnI fragment was cloned between the PstI and KpnI sites of pSH19, resulting in pSH19-srsA.HPLC Analysis of Phenolic Lipids Produced by Streptomyces—S. lividans TK21 harboring pFM1, pFM2, or pFM3 was inoculated to 100 ml of yeast extract-malt extract liquid medium containing 5 μg/ml of kanamycin and grown at 30 °C. After 24 h, 5 μg/ml of thiostrepton was added to induce the tip promoter, and the culture was continued for further 48 h. A portion of the culture broth was adjusted to pH 1.0 with 6 m HCl and extracted with ethyl acetate. After evaporation to dryness, the residue was dissolved in 10 μl of dimethyl sulfoxide for reversed phase HPLC analysis. The conditions for analytical HPLC were: ODS-80Ts column (4.6 × 150 mm; Tosoh, Tokyo) eluted isocratically with 95% CH3CN and 0.1% trifluoroacetic acid in water at a flow rate of 0.8 ml/min. UV absorbance was detected at 280 nm.Large Scale Preparation and Characterization of Phenolic Lipids—The culture conditions of S. lividans TK21 harboring pFM1 or pFM2 and S. griseus harboring pFM1 were the same as those for the analytical scale, except that the culture was scaled up to 1 liter × 3. After cultivation, the mycelia obtained by centrifugation were extracted with chloroform/methanol (2:1, v/v), the cell debris was removed by filtration, and the filtrate was lyophilized to dryness. The culture broth was adjusted to pH 1.0 with 6 m HCl and then extracted with hexane. The hexane extract was passed through a pad of Celite to remove emulsions. The Celite was washed once with hexane. The organic layers were combined, washed with brine, dried with Na2SO4, and evaporated to dryness. The crude materials from the mycelia and the culture broth were dissolved in a small amount of chloroform/methanol (1:1, v/v) and flash-chromatographed on silica gel using chloroform/ethyl acetate (85:15, v/v) as an eluent. The eluate was evaporated and dissolved in methanol for reversed phase preparative HPLC. Compounds 1-17 were purified by reversed phase preparative HPLC equipped with a Pegasil C4 column (10 × 250 mm; Senshu Scientific, Tokyo) by elution isocratically with 95% CH3CN and 1% acetic acid in water at a flow rate of 3 ml/min. The collected fractions were lyophilized to give 1-17 as white solids. From S. lividans TK21 harboring pFM1, 4 mg of 1, 7 mg of a mixture of 2, 3, and 4, 21 mg of 5, and 33 mg of a mixture of 6 and 7 were isolated. From S. lividans TK21 harboring pFM2, 4 mg of 8, 14 mg of 9, and 20 mg of a mixture of 10 and 11 were isolated. From S. lividans TK21 harboring pFM3, 5 mg of a mixture of 9 and 15, 1 mg of a mixture of 10, 11, 16, and 17, 6 mg of 12, and 4 mg of a mixture of 13 and 14 were isolated. The structure of each phenolic lipid was determined by proton and carbon NMR spectroscopy, with the aid of heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation analysis, and LC-APCIMS and LC-APCIMS/MS analysis. The NMR data are summarized in the supplemental text. The MS data are summarized in supplemental Table S1.Production and Purification of SrsA—S. lividans TK21 harboring pSH19-srsA was inoculated to 2 liters of yeast extractmalt extract liquid medium containing 5 μg/ml thiostrepton and grown at 30 °C. After 48 h, ϵ-caprolactam (final concentration, 0.1% w/v) was added to induce the nitA promoter, and the culture was continued for a further 60 h. Mycelium was harvested by centrifugation and resuspended in 50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, and 10 mm imidazole. After sonication, cell debris was removed by centrifugation. The cleared lysate was applied to nickel-nitrilotriacetic acid spin columns (Qiagen), washed five times with 50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, and 80 mm imidazole, and eluted with 50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, and 250 mm imidazole. The purified SrsA (∼1.0 mg) was dialyzed twice against 2 liters of 10 mm Tris-HCl (pH 8.0). Protein concentration was measured with a Bio-Rad protein assay kit using bovine serum albumin as a standard. Purity of the recombinant SrsA was checked by SDS-polyacrylamide gel electrophoresis (supplemental Fig. S1).In Vitro SrsA Reaction—14-Methylpentadecanoyl-CoA and 14-methylhexadecanoyl-CoA were prepared according to the method of Blecher (9Blecher M. Methods Enzymol. 1981; 72: 404-408Crossref PubMed Scopus (23) Google Scholar). The reactions, containing 100 mm Tris-HCl (pH 7.5), 29 μg of SrsA, 200 μm starter-CoA, and 100 μm extender-CoA(s), were performed in a total volume of 300 μl. The reactions were incubated at 30 °C for 3 h before being quenched by 60 μl of 6 n HCl and extracted with 300 μl of ethyl acetate. The organic layer was evaporated, and the residue dissolved in 30 μl of methanol for HPLC analysis. The conditions for analytical HPLC were as described above.Gene Disruption of srsA in S. griseus—An in-frame deletion in the chromosomal srsA gene was introduced by replacing the region encoding from Gly-33 to Leu-302 with a SalI recognition sequence of six nucleotides (see Fig. 1). A 1.5-kb region upstream from the Pro-32 codon was amplified by using primer XI (5′-GCGGAATTCCTTGCCGTCGTTGGCGTTGG-3′; with an EcoRI site shown by the italic letters) and primer XII (5′-GCGGTCGACGGGCGGCAGGCAGGTGCGGG-3′; with the Pro-32 codon shown by the boldface letters and a SalI site shown by the italic letters). The amplified fragment was cloned between the EcoRI and SalI sites of pUC19, resulting in pUC-ΔsrsAN. A 1.5-kb region downstream from the Ala-303 codon was amplified by using primer XIII (5′-GCGGTCGACGCCGACGTCGGCAACCTG-3′; with the Ala-303 codon shown by the boldface letters and a SalI site shown by the italic letters) and primer XIV (5′-GCGGACGTCGCGATGCCCTCCCCGG-3′; with a PstI site shown by the italic letters). The amplified fragment was cloned between the SalI and PstI sites of pUC19, resulting in pUC-ΔsrsAC. The SalI-PstI fragment excised from the pUC19-ΔsrsAC was cloned between the SalI and PstI sites of pUC19-ΔsrsAN, resulting in pUC19-ΔsrsA. The kanamycin resistance gene (aphII) from Tn5 (10Beck E. Ludwig G. Auerswald E.A. Reiss B. Schaller H. Gene (Amst.). 1982; 19: 327-336Crossref PubMed Scopus (694) Google Scholar) was inserted in the HindIII site of the pUC19-ΔsrsA, resulting in pUC19-ΔsrsAK. pUC19-ΔsrsAK was denatured with NaOH and introduced by protoplast transformation into S. griseus IFO13350. Transformants containing pUC19-ΔsrsAK in the chromosome as a result of single cross-over were selected among kanamycin (10 μg/ml)-resistant colonies. One of the kanamycin-resistant colonies was grown for a week on YMPD agar without kanamycin. Spores recovered were spread on YMPD agar without kanamycin. From these colonies, one ΔsrsA mutant, in which chromosomal srsA was in-frame deleted, was isolated as a kanamycin-sensitive colony. The correct replacement was confirmed by Southern hybridization. The phenolic lipids were extracted from mutant ΔsrsA that had been grown at 28 °C for 4 days in YMPD medium.Subcellular Distribution of Phenolic Lipids in S. griseus—We fractionated the culture into the extracellular, cell wall, cell membrane, and cytoplasmic fractions, according to the method of Pertiwiningrum et al. (11Pertiwiningrum A. Ino Y. Suzuki T. Iwama T. Kawai K. J. Biosci. Bioeng. 2004; 98: 214-216Crossref PubMed Scopus (3) Google Scholar). The wild-type strain S. griseus IFO13350 and mutant ΔsrsA were grown in 50 ml of liquid YMPD medium at 30 °C for 48 h. The cells were collected from 5 ml of culture by centrifugation at 10,000 × g. The supernatant was collected to prepare the extracellular fraction. The cells were washed with 5 ml of Tris-HCl (pH 7.5) containing 0.4 m sucrose and suspended in 5 ml of Tris-HCl (pH 7.5) containing 0.4 m sucrose and 1 mg/ml lysozyme. The mixture was incubated at 37 °C for 1 h to prepare spheroplasts. The formation of spheroplasts was checked by light microscopic observation. The spheroplasts were collected by centrifugation at 4,000 × g. The supernatant was collected to prepare the cell wall fraction. The spheroplasts were washed with 5 ml of Tris-HCl (pH 7.5) containing 0.4 m sucrose, and then 3 ml of water was added to burst the spheroplasts. The suspension was centrifuged at 100,000 × g for 1 h. The supernatant was the cytoplasmic fraction, and the precipitate was the membrane fraction. All of the fractions were acidified with 1 m HCl and extracted with ethyl acetate. The organic layer was evaporated, and the residue was dissolved in 20 μl of methanol for HPLC analysis. The conditions for analytical HPLC were as described above.RESULTSOrganization of the srs Operon and Homology Search of Srs Proteins—The gene organization of the 3-kb srs genes is shown in Fig. 1. The stop codon of srsA is 13 nucleotides upstream from the start codon of srsB, and the TGA stop codon of srsB is overlapped with the GTG start codon of srsC. The organization of the srs genes suggested that these three formed an operon that was transcribed from a promoter upstream of srsA. orf1, encoding a protein weakly homologous to a prenyltransferase UbiA (12Siebert M. Bechthold A. Melzer M. May U. Berger U. Schröder G. Schröder J. Severin K. Heide L. FEBS Lett. 1992; 307: 347-350Crossref PubMed Scopus (60) Google Scholar), which is located 118 nucleotides upstream of srsA or orf2, encoding a putative transmembrane efflux protein, which is located 217 nucleotides downstream of srsC, that appeared not to be functionally linked to the srs operon. We did not study orf1 or orf2 further because the srs operon was sufficient for production of phenolic lipids in S. lividans (see below).SrsA, consisting of 350 amino acid residues, shared 61% amino acid sequence identity to pks10 that is annotated as a functionally unknown type III PKS and is widely distributed among mycobacteria. SrsA also showed 57% identity to pks11 in Mycobacterium tuberculosis (supplemental Fig. S2), a type III PKS that catalyzes α-pyrone synthesis from long chain aliphatic acyl-CoA substrates (13Saxena P. Yadav G. Mohanty D. Gokhale R.S. J. Biol. Chem. 2003; 278: 44780-44790Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The similarity of SrsA to pks11 did not necessarily predict the catalytic property of SrsA, because the properties of pks11 were determined only by in vitro reaction and because type III PKSs are promiscuous with respect to substrate specificity and produce α-pyrones from various starter molecules (1Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (712) Google Scholar).A BLAST search revealed that SrsB, consisting of 177 amino acid residues, contained an isoprenylcysteine carboxyl methyltransferase domain of the isoprenylcysteine carboxyl methyltransferase family members that are unique membrane proteins involved in post-translational modification of oncogenic proteins (14Winter-Vann A.M. Casey P.J. Nat. Rev. 2005; 5: 405-412Crossref Scopus (274) Google Scholar). Because srsB is conserved in various bacterial species and, in most cases, consists of an operon with a type III PKS gene (supplemental Fig. S3), we assumed that SrsB was concerned with a modification of the polyketides produced by SrsA. Sequence alignment of SrsB with Ste14p (15Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar), an isoprenylcysteine carboxyl methyltransferase family protein in Saccharomyces cerevisiae, along with a computational search for secondary structure prediction of membrane proteins, predicted that SrsB possessed two transmembrane helices (supplemental Fig. S4).SrsC, consisting of 338 amino acid residues, contained a sequence motif found in flavoprotein hydroxylases (16Enroth C. Neujahr H. Schneider G. Lindqvist Y. Structure. 1998; 6: 605-617Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (supplemental Fig. S5), suggesting that SrsC could be a FAD-dependent hydroxylase. srsC is conserved among actinomycetes as a member of the operons including srsA and srsB in the same order (supplemental Fig. S3).Analysis of Lipids Produced by S. lividans Overexpressing srs Genes—We constructed three plasmids, pFM1 carrying srsA, pFM2 carrying srsA and srsB, and pFM3 carrying srsA, srsB, and srsC and introduced them into a heterologous host, S. lividans TK21 (Fig. 1). The srs genes were under the strong, thiostrepton-inducible tipA promoter on the high copy number plasmid pIJ6021 for overexpression of the srs genes. We analyzed extracts from culture broths and mycelium by HPLC, on the assumption that phenolic lipids might be produced in large amounts and leaked in the culture broth in detectable amounts. Under the conditions employed, S. lividans harboring only the empty vector produced no detectable phenolic lipids (Fig. 2A). On the other hand, the recombinant S. lividans strains harboring the srs genes produced several lipids, giving multiple peaks on HPLC (Fig. 2, B-D).FIGURE 2HPLC chromatograms and the structures of phenolic lipids isolated from recombinant S. lividans TK21 carrying a combination of the srs genes. HPLC chromatograms of the lipid fraction prepared from the culture broth of S. lividans TK21 harboring the empty vector pIJ6021 (A), pFM1 carrying srsA (B), pFM2 carrying srsA and srsB (C), and pFM3 carrying srsA, srsB, and srsC (D) are shown. The iso- and anteiso-isomers (2 + 3, 6 + 7, 10 + 11, 13 + 14, and 16 + 17) could not be separated. The second peak in B contained 4 and mixture X, as revealed with a preparative-scale column. Mixture X was composed of at least 2 and/or 3 and a monounsaturated form(s) of 6 and/or 7. The peak containing compound 8 in (C) also contained very small amounts of mono-unsaturated forms of 10 + 11. The third peak in (D) contained 15 and a very small amount of 9. The fourth peak in D contained compounds 16 + 17 and very small amounts of 10 + 11. The structures of the phenolic lipids 1-19 are shown in E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For structural determination by NMR analysis of the lipids that were contained in the peaks in Fig. 2, lipids were separated by HPLC equipped with a preparative C4 column. As a result, we separated lipids in a total of 17. We determined their chemical structures (Fig. 2E) by proton and carbon NMR (supplemental text), with the aid of heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation analysis (data not shown), and LC-APCIMS and LC-APCIMS/MS analysis (supplemental Table S1). S. lividans harboring pFM1 produced alkylresorcinols (compounds 4-7) as major products and an alkylpyrone (compound 1) as a minor product. SrsA was therefore a type III PKS responsible for phenolic lipid synthesis from the substrates produced by the wild-type S. lividans strain.On the other hand, S. lividans harboring pMF2 produced alkylresorcinol methyl ethers (compounds 8-11), suggesting that SrsB was a methyltransferase acting on alkylresorcinols. In fact, alkylpyrones 1-3 remained unmethylated," @default.
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• W2017109849 title "Phenolic Lipids Synthesized by Type III Polyketide Synthase Confer Penicillin Resistance on Streptomyces griseus" @default.
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