Characterization of the Azinomycin B Biosynthetic. Gene Cluster Revealing a Different Iterative Type I

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1 Chemistry & Biology 15 Supplemental Data Characterization of the Azinomycin B Biosynthetic Gene Cluster Revealing a Different Iterative Type I Polyketide Synthase for Naphthoate Biosynthesis Qunfei Zhao, Qingli He, Wei Ding, Mancheng Tang, Qianjin Kang, Yi Yu, Wei Deng, Qi Zhang, Jie Fang, Gongli Tang, and Wen Liu Bacterial Strains and Plasmids Table S1. Bacterial Strains and Plasmids Used in This Study Strain/Plasmid Characteristic(s) Reference E. coli DH5α Host for general cloning Invitrogen LE392 Host for constructing the genomic library Promega S17-1 Donor strain for conjugation between E.coli Mazodier et al., 1989 and Streptomyces S. sahachiroi NRRL 2485 Wild type strain, Azinomycin B producing NRRL AL1001 ΔaziB mutant, Azinomycin B non-producing AL1002 ΔaziA3 mutant, Azinomycin B non-producing AL1003 ΔaziC7 mutant, Azinomycin B non-producing 1

2 S. albus J1074 Host for heterologous expression Chater et al., 1980 AL1004 J1074 containing pal1012, expression of AziB alone, 5-methy-NPA producing AL1005 J1074 containing pal1019, expression of AziB, AziB1 and AziB2, 3-methoxy-5-methyl-NPA producing AL1006 J1074 containing the vector ptgv2, negative control AL1007 J1074 containing pal1027, expression of the KR mutant AziB (G1398A), 5-methy-NPA non-producing AL1008 J1074 containing pal1028, expression of the KR mutant AziB (Y1549F), 5-methy-NPA non-producing AL1009 J1074 containing pal1028, expression of the DH mutant AziB (H935F), 5-methy-NPA non-producing Plasmids psp72 E. coli subcloning vector Promega pgem -7zf E. coli subcloning vector Promega pant841 E. coli subcloning vector GenBank (accession No. 2

3 AF438749) pbluescript SK+ E. coli subcloning vector Stratagene pkc1139 E.coli-Streptomyces shuttle vector for gene Bierman et al., 1992 inactivation, temperature sensitive replication in Streptomyces ptgv2 E.coli-Streptomyces shuttle vector for Tang, unpublished heterologous expression pal1007 psp72 derivative containing a 0.9 kb PCR product of azib (P1) pal1008 pkc1139 derivative containing a 2.8 kb internal fragment of azib, construct for azib inactivation pal1009 pant841 derivative containing a 1.1 kb internal fragment of azia3 pal1010 pkc1139 derivative containing a 1.1 kb internal fragment of azia3, construct for azia3 inactivation pal1011 pkc1139 derivative containing a mutant azic7, in which a 839 bp internal fragment was replaced by the erythromycin resistance gene (erye), construct for azic7 inactivation pal1013 psp72 derivative that contains a 1.5 kb PCR 3

4 product encoding the KS domain of AziB pal1014 psp72 derivative that contains a 1.2 kb PCR product encoding the AT domain of AziB pal1017 psp72 derivative that contains a 3.2 kb fragment encoding the KS-AT domain of AziB under the control of the PermE* promoter pal1015 psp72 derivative that contains a 1.0 kb PCR product encoding the DH domain of AziB pal1016 psp72 derivative that contains a 1.9 kb PCR product encoding the KR-ACP domain of AziB pal1018 psp72 derivative that contains a 2.9 kb fragment encoding the DH-KR-ACP domain of AziB pal1012 ptgv2 derivative, construct carrying azib alone under the control of PermE* promoter pal1027 ptgv2 derivative, construct carrying the azib derivative encoding a KR mutant protein (G1398A) pal1028 ptgv2 derivative, construct carrying the azib derivative encoding a KR mutant protein 4

5 (Y1549F) pal1029 ptgv2 derivative, construct carrying the azib derivative encoding a DH mutant protein (H935F) pal1020 psp72 derivative that contains a 2.7 kb fragment carrying azib1 and azib2 pal1021 pgem-7zf derivative that contains a 3.1 kb fragment carrying azib1 and azib2 under the control of PermE* promoter pal1019 ptgv2 derivative, construct carrying azib, azib1 and azib2 with the PermE*-aziB + PermE*-aziB1-aziB2 genotype pal1022 S. sahachiroi NRRL 2485 genomic library cosmid pal1023 S. sahachiroi NRRL 2485 genomic library cosmid pal1024 S. sahachiroi NRRL 2485 genomic library cosmid pal1025 S. sahachiroi NRRL 2485 genomic library cosmid pal1026 S. sahachiroi NRRL 2485 genomic library cosmid 5

6 Gene Inactivation in S. sahachiroi To inactivate the iterative type I PKS gene azib, a 2.8 kb BamHI internal fragment of azib from cosmid pal1023 was directly cloned into pkc1139, yielding the recombinant construct pal1008. To inactivate the NRPS gene azia3, a 1.1 kb BamHI/NcoI internal fragment from cosmid pal1024 was cloned into pant841 to make pal1009. This fragment was then recovered from pal1009 and inserted into the BamHI/HindIII site of pkc1139, yielding the recombinant construct pal1010. To inactivate the aminotransferase gene azic7, a 2.0 kb HindIII/XbalI fragment amplified by PCR using the primers 5 -TA TAT AAG CTT CAG CCC GGC GCG TAC CTT G-3 (HindIII site underlined) and 5 -ATA TCT AGA TGG GGG TGT GCC CTT GTG GAG-3 (XbaI site underlined) and a 2.0 kb EcoRV/EcoRI fragment amplified by PCR using the primers 5 -AT GAT ATC CCG GGG CTC GCA GCT ACA TC-3 (EcoRV site underlined) and 5 -AT GAA TTC CCG GGC ACC TCG TGC AAC TG-3 (EcoRI site underlined), were co-ligated with the erythromycin resistance gene, erye. The resultant 5.9 kb HindIII/EcoRI fragment was cloned into the same site of pkc1139 to generate the recombinant construct pal1011, in which the 839 bp fragment of azic7 was deleted and replaced by erye. Introduction of plasmid DNA into S. sahachiroi was carried out by E. coli-streptomyces conjugation, following the procedure described previously (Liu and Shen, 2000). For gene disruption, colonies that were apramycin resistant at 37 C were identified as the mutants. For gene replacement, colonies that were erythromycin resistant and apramycin sensitive at 37 C were identified as the mutants. 6

7 Heterogenous Expression in S. albus To make the azib expression construct, the DNA fragments respectively encoding individual functional domains of AziB were amplified by PCR and assembled by using a multiple-fragment ligation strategy. A 1.5 kb PCR product encoding the KS domain was obtained by using the primers 5 -AT GAA TTC TCT AGA GCG ACT GCC TGG CAC-3 (XbaI site underlined) and 5 -TA TAT AAG CTT AAG GGG AAG ACC TGG GGG CG-3 (AflII site underlined) and cloned into psp72, yielding pal1013; A 1.2 kb PCR product encoding the AT domain was obtained by using the primers 5 -TA GAA TTC CTT AAG CGC CGC CTC CGA GCAG-3 (AflII site underlined) and 5 -TA TAT AAG CTT AGA TCT CCG TCG TAG GAC CAG GTG-3 (HindIII and BglII site underlined) and cloned into psp72, yielding pal1014. These 1.5 kb XbaI/AflII and 1.2 kb HindIII/AflII fragments were respectively recovered from pal1013 and pal1014 and then ligated with a 0.5 kb EcoRI/XbaI fragment containing the PermE* promoter in psp72, yielding pal1017, in which the 2.7 kb fragment encoding the N-terminal KS-AT domain of AziB is under the control of the PermE* promoter. On the other hand, a 1.0 kb PCR product encoding the DH domain was obtained by using the primers 5 -TA GAA TTC AGA TCT CGT GGA CGT CCC CGC-3 (EcoRI and BglII site underlined) and 5 -TA TAT AAG CTT CCT AGG GCC GAC CGC GCC G-3 (AvrII site underlined) and cloned into psp72, yielding pal1015; A 1.9 kb fragment encoding the KR-ACP domain was obtained using the primers 5 -TA GAA TTC CCT AGG GAC CTG GTG CAC GAA CTC-3 (AvrII site underlined) and 5 -TA TAT AAG CTT CTA TAC TGC GAC GAC CTT CTG-3 (HindIII site underlined) and cloned into psp72, yielding pal1016. These 1.0 kb EcoRI/AvrII and 1.8 kb AvrII/HindIII fragments were respectively recovered from pal1015 and pal1016 and ligated into psp72, yielding pal1018 that contains the 2.9 kb fragment encoding the 7

8 C-terminal DH-KR-ACP domain of AziB. Finally, these 3.2 kb EcoRI/BglII fragment from pal1017 and 2.9 kb BglII/HindIII fragment from pal1018 were recovered and cloned into ptgv2, yielding the recombinant construct pal1012 in which azib alone is under the control of the PermE* promoter. To inactivate the KR domain of AziB, with pal1016 that contains a 1.9 kb fragment encoding the KR-ACP domain as the template, PCR amplifications were carried out using the primers 5 -C ACC GGC GGT CTG GCC GCG CTC GGC CTG G-3 and 5 -C CAG GCC GAG CGC GGC CAG ACC GCC GGT G-3 for mutation of G1398 to A within the NADPH-binding motif, and the primers 5 -CC GGC CAG GTC AGC TTC GCC TCC GCG AAC TC-3 and 5 -GA GTT CGC GGA GGC GAA GCT GAC CTG GCC GG-3 for mutation of Y1549 to F at the conserved active site. To inactivate the DH domain of AziB1, with pal1015 that contains a 1.0 kb fragment encoding the DH domain as the template, PCR amplification was carried out using the primers 5 -CCG TAC GCG CAG TCG TTC AAG GTC GTC GGC GTG-3 and 5 -CAC GCC GAC GAC CTT GAA CGA CTG CGC GTA CGG-3 for mutation of H935 to F at the conserved active site. Using the similar multiple-fragment ligation strategy described above, these mutant DNA fragments were assembled with the fragments encoding other functional domains, yielding the recombinant constructs pal1027 for expressing the KR mutant AziB (G1398A), pal1028 for expressing the KR mutant AziB (Y1549F) and pal1029 for expressing the DH mutant AziB (H935F), respectively. To make the azib, azib1 and azib2 co-expression construct, a 2.7 kb PstI/SphI fragment from cosmid pal1026 was cloned into psp72, yielding pal1020 that contains azib1 and azib2. This 2.7 kb XbaI/HindIII fragment was recovered and ligated with a 0.5 kb EcoRI/XbaI PermE* 8

9 promoter in pgem-7zf, yielding pal1021, in which both genes are under the control of this promoter. Next, The 3.1 kb XhoI/HindIII fragment from pal1021 and the 6.1 kb EcoRI/XhoI fragment from pal1012 were respectively recovered and cloned into ptgv2, yielding the recombinant construct pal1019 with the genotype PermE*-aziB + PermE*-aziB1-aziB2 for heterologous expression of all three proteins together. Introduction of plasmid DNA into S. albus was carried out by E. coli-streptomyces conjugation, following the procedure described previously (Liu and Shen, 2000) Colonies that were Thiostrepton resistant were identified as the recombinant strains. Tandem Mass Spectrometry Analysis of Azinomycin B Tandem Mass Spectrometry analysis of azinomycin B was carried on a LCQ Fleet Ion Trap MSn tandem mass spectrometer (Thermo Fisher Scientific). 9

10 Figure S1. MS Spectrum of Azinomycin B [M + H] + Ion at m/z = (A), and MS/MS Spectrum of the Parent Ion at m/z = (B) A B 10

11 Synthesis of 3-methoxy-5-methyl-NPA Figure S2. Synthesis of 3-methoxy-5-methyl-NPA Br 1) Mg, THF, reflux O 2), THF, 0 3) NH 4 Cl DMSO, (COCl) 2 OH Et 3 N, CH 2 Cl 2, -78 EtONa, EtOH (CO 2 Et) 2 O O O OEt OH H 2 SO 4, CHCl 3 0 rt OH NaH, CH 3 I THF, rt O CO 2 Et CO 2 Et LiOH, rt CH 3 OH, H 2 O CO 2 H O Synthesis of 3-methoxy-5-methyl-NPA (illustrated in Figure S2) was achieved by following the previously described method (Helen et al., 1998). The 1 H NMR and 13 C NMR spectra were measured on a Bruker AVANCE500 (500 MHz) spectrometer. 1 H NMR (500 MHz, CDCl 3 ) δ: 2.69 (s, 3H), 3.99 (s, 3H), 7.36~7.41(m, 2H), 7.54 (d, 1H, J = 2.6 Hz), 8.04 (d, 1H, J = 2.6 Hz), and 8.80 (brd, 1H, J = 7.9 Hz). 13 C NMR(125MHz, CDCl 3 ) δ : 20.2 (CH 3 ), 55.6 (CH 3 ), (CH), (CH), (CH), (CH), (C), (C), (C), (C), (C), (C), and (C). Analysis of 3-thoxy-5-thyl-NPA Produced in S. sahachiroi High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of 3-methoxy-5-methyl-NPA was carried out on an Agilent Rp18 column ( mm, part No , serial No. USCL021611). The column was equilibrated with 50% solvent A (H 2 O) 11

12 and B (CH 3 CN), and developed with the following program: 0-5 min, a linear gradient from 90% A/10% B to 80% A/20% B; 5-25 min, a linear gradient from 80% A/20% B to 30% A/70% B; min, a linear gradient from 30% A/70% B to 5% A/95% B; min, constant 5% A/95% B; and min, a linear gradient from 5% A/95% B to 90% A/10% B. This was carried out at a flow rate of 0.5 ml/min and UV detection at 220 nm using an Agilent 1100 HPLC system (Agilent Technologies. Palo Alto, CA). Figure S3. HPLC Analysis of 3-thoxy-5-thyl-NPA Produced in Various Strains: (I) Synthetic Standard (Control), (II) S. sahachiroi Wild-Type Strain; (III) AL1001; (IV) AL1003; and (V) AL1002 Solid asterisk indicates 3-methoxy-5-methyl-NPA. Sequence Analysis Figure S4. Multiple Sequences Alignment of KR Domains from Various Iterative Type I PKSs, 12

13 Including Those for Aromatic Polyketide Biosynthesis, AziB, NcsB (AAM77986), ChlB1 (AAZ77673) and MdpB (ABY66019), and MchA (CAG28678) for Aliphatic Chain Formation, with That from the Noniterative Type I PKS WcbR (YP ) as a Control The KR sequence of AziB is boxed. The conserved amino acids with asterisks indicate the key residues at the catalytic region. The KR domain of AziB (similar to those of NcsB, ChlB1, MdpB, and MchA) contains the conserved residues of K, S, Q, Y and N at the catalytic region (Reid et al., 2003; Caffrey, 2003; Wu et al., 2005), suggesting that this KR domain is active to reduce the keto group. anwhile, the KR domain of AziB lacks the H residue at the active site groove and characteristic L-D-D motif (labeled with fillet rectangle), implicating that it falls into a A1 type KR family and acts on the keto groups to yield L-hydroxy configurations exclusively (Keatinge-Clay, 2007). Supplemental References 1. Bierman, M., Logan, R., O Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, Caffrey, P. (2003) Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases. ChemBioChem 4,

14 3. Chater K.F., and Wilde L.C. (1980). Streptomyces albus G mutants defective in the SalGI restriction-modification system. J. Gen. Microbiol. 116, Helen J.B., Christophe Y. D., Timothy J. H., Michael B. H., K. M. A., and Michael S. (1998). Asymmetric synthesis of the left hand portion of the azinomycins. J. Chem. Soc., Perkin Trans. 1, Keatinge-Clay, A. T. (2007). A Tylosin Ketoreductase Reveals How Chirality Is Determined in Polyketides. Chem. Biol. 14, Liu, W., and Shen, B. (2000). Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the caga gene that encodes the C-1027 apoprotein. Antimicrob. Agents. Chemother. 44, Mazodier, H., Petter, R., and Thompson, C. (1989). Intergeneric conjugation between Escherichia and Streptomyces species. J. Bacteriol. 171, Reid, R., Piagentini, M., Rodriguez, E., Ashley, G., Viswanathan, N., Carney, J., Santi, D.V., Hutchinson, C.R., and McDaniel, R. (2003). A model of structure and catalysis for ketoreductase domains in modular polyketide synthases, Biochemistry 42, Wu, J., Zaleski, T.J., Valenzano, C., Khosla, C., and Cane, D.E. (2005) Polyketide double bond biosynthesis. chanistic analysis of the dehydratase-containing module 2 of picromycin/methymycin polyketide synthase. J. Am. Chem. Soc. 127,