Salinispora Arenicola CNP193新颖NRPS基因簇在Streptomycetes Coelicolor M512中的异源表达
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摘要
异源表达海洋专性放线菌salinispora arenicola CNP193新颖NRPS基因簇。PCR扩增基因簇两端各约1 000 bp的片段,融合PCR将两片段融合,同质粒pC AP01双酶切后连接,构建基因簇捕获载体pC AP01-preP KS/NRPS,通过TAR(transformation-associated recombination)克隆技术构建基因簇捕获质粒pC AP01-PKS/NRPS,运用三亲接合转移法将pC AP01-PKS/NRPS转化streptomycetes coelicolor M512。HPLC检测对照菌株和重组菌发酵液乙酸乙酯提取物初步表明基因簇有表达产物产生。为新颖salinispora arenicola CNP193新颖NRPS基因簇的鉴定奠定基础。
Heterologous expression the novel NRPS gene cluster mined from obligate marine actinomycete strain salinispora arenicola in streptomycetes coelicolor M512. The two 1. 0-kb regions corresponding to the left and the right end of the tar gene cluster were PCR,respectively. The two PCR produces were then combined and assembled into single piece. The assembled fragment was digested with Spe I and Kpn I and introduced into p CAP01. Direct TAR cloning of the gene cluster from genomic DNA was carried out,and the resulting plasmid was transferred to S.coelicolor M512 by triparental intergeneric conjugation facilitated by E. coli ET12567 / p UB307. The product of the gene cluster was detected through analyzed the ethyl acetate extracts from the fermentation broth of recombinant and CK strains by reversed phase HPLC. The results lay the foundation for elucidation the pathway of the novel NRPS gene cluster mined from obligate marine actinomycete strain salinispora arenicola CNP193.
Heterologous expression the novel NRPS gene cluster mined from obligate marine actinomycete strain salinispora arenicola in streptomycetes coelicolor M512. The two 1. 0-kb regions corresponding to the left and the right end of the tar gene cluster were PCR,respectively. The two PCR produces were then combined and assembled into single piece. The assembled fragment was digested with Spe I and Kpn I and introduced into p CAP01. Direct TAR cloning of the gene cluster from genomic DNA was carried out,and the resulting plasmid was transferred to S.coelicolor M512 by triparental intergeneric conjugation facilitated by E. coli ET12567 / p UB307. The product of the gene cluster was detected through analyzed the ethyl acetate extracts from the fermentation broth of recombinant and CK strains by reversed phase HPLC. The results lay the foundation for elucidation the pathway of the novel NRPS gene cluster mined from obligate marine actinomycete strain salinispora arenicola CNP193.
引文
1 Lanea A L,Moore B S.A sea of biosynthesis:marine natural products meet the mmolecular age.Natural Product Reports,2011;28(2):411-428
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5 Jensen P R,Chavarria K L,Fenical W,et al.Challenges and triumphs to genomics-based natural product discovery.Journal of Industrial Microbiology&Biotechnology,2014;41(2):203-209
6 Ziemert N,Podell S,Penn K,et al.The natural product domain seeker NaP DoS:a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity.PloS One,2012;(7):e34064
7房耀维,刘姝,王淑军,等.海洋放线菌盐孢菌属研究进展.微生物学通报,2014;41(6):1203-1210Fang Yaowei,Liu Shu,Wang Shujun,et al.Recent advance in the marine genus salinispora.Microbiology China,2014;41(6):1203-1210
8 Wilkinson B,Micklefield J.Mining and engineering natural-product biosynthetic pathways.Nature Chemical Biology,2007;3(7):379-386
9朱梦奕,何璟.放线菌基因组大片段克隆载体及异源表达宿主改造的研究进展.微生物学通报,2013;40(10):1920-1928Zhu Mengyi,He Jing.Cloning vectors and heterologous hosts for large fragments of actinobacteria genomic DNA.Microbiology China,2013;40(10):1920-1928
10 Komatsu M,Uchiyama T,Omura S,et al.Genome-minimized streptomyces host for the heterologous expression of secondary metabolism.Proceedings of the National Academy of Sciences of the United States of America,2010;107(6):2646-2651
11 Xue Y,Sherman D H.Alternative modular polyketide synthase expression controls macrolactone structure.Nature,2000;403(3):571-575
12 Yamanaka K,Reynolds K A,Kersten R D,et al.Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A.Proceedings of the National Academy of Sciences of the United States of America,2014;(11):1957-1962
13 Crawford J M,Clardy J.Microbial genome mining answers longstanding biosynthetic questions.Proceedings of the National Academy of Sciences of the United States of America,2012;109(20):7589-7590.
14 Xue Y,Sherman D H.Alternative modular polyketide synthase expression controls macrolactone structure.Nature,2000;403(3):571-575
15 Gomez-Escribano J P,Bibb M J.Streptomyces coelicolor as an expression host for heterologous gene clusters.Methods in Enzymology,2012;517:279-300
16 Baltz R H.Streptomyces and saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters.Journal of Industrial Microbiology and Biotechnology,2010;37(8):759-772
2 Obrien R V,Davis R W,Khosla C,et al.Computational identification and analysis of orphan assembly-line polyketide synthases.The Journal of Antibiotics,2014;67(1):89-97
3姚琳,郭东林.PKS-NRPS数据库的研究进展.中国农学通报,2009;25(16):280-283Yao Lin,Guo Donglin.Advances in PKS-NRPS databases.Chinese Agricultural Science Bulletin,2009;25(16):280-283
4 Lane A L,Moore B S.A sea of biosynthesis:marine natural products meet the molecular age.Natural Product Reports,2011;28(2):411-428
5 Jensen P R,Chavarria K L,Fenical W,et al.Challenges and triumphs to genomics-based natural product discovery.Journal of Industrial Microbiology&Biotechnology,2014;41(2):203-209
6 Ziemert N,Podell S,Penn K,et al.The natural product domain seeker NaP DoS:a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity.PloS One,2012;(7):e34064
7房耀维,刘姝,王淑军,等.海洋放线菌盐孢菌属研究进展.微生物学通报,2014;41(6):1203-1210Fang Yaowei,Liu Shu,Wang Shujun,et al.Recent advance in the marine genus salinispora.Microbiology China,2014;41(6):1203-1210
8 Wilkinson B,Micklefield J.Mining and engineering natural-product biosynthetic pathways.Nature Chemical Biology,2007;3(7):379-386
9朱梦奕,何璟.放线菌基因组大片段克隆载体及异源表达宿主改造的研究进展.微生物学通报,2013;40(10):1920-1928Zhu Mengyi,He Jing.Cloning vectors and heterologous hosts for large fragments of actinobacteria genomic DNA.Microbiology China,2013;40(10):1920-1928
10 Komatsu M,Uchiyama T,Omura S,et al.Genome-minimized streptomyces host for the heterologous expression of secondary metabolism.Proceedings of the National Academy of Sciences of the United States of America,2010;107(6):2646-2651
11 Xue Y,Sherman D H.Alternative modular polyketide synthase expression controls macrolactone structure.Nature,2000;403(3):571-575
12 Yamanaka K,Reynolds K A,Kersten R D,et al.Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A.Proceedings of the National Academy of Sciences of the United States of America,2014;(11):1957-1962
13 Crawford J M,Clardy J.Microbial genome mining answers longstanding biosynthetic questions.Proceedings of the National Academy of Sciences of the United States of America,2012;109(20):7589-7590.
14 Xue Y,Sherman D H.Alternative modular polyketide synthase expression controls macrolactone structure.Nature,2000;403(3):571-575
15 Gomez-Escribano J P,Bibb M J.Streptomyces coelicolor as an expression host for heterologous gene clusters.Methods in Enzymology,2012;517:279-300
16 Baltz R H.Streptomyces and saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters.Journal of Industrial Microbiology and Biotechnology,2010;37(8):759-772