Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

Danfeng Song

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

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Jane Coughlin,

Jane Coughlin

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

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Jianhua Ju,

Jianhua Ju

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

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Xiufen Zhou,

Xiufen Zhou

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

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Ben Shen,

Ben Shen

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

National Cooperative Drug Discovery Group of University of Wisconsin, University of Wisconsin, Madison, Wisconsin 53705, USA

Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705, USA

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Chunhua Zhao,

Corresponding Author

Chunhua Zhao

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

*Corresponding authors: Zinxin Deng: Tel, 86-21-62933404; E-mail, [email protected] Chunhua Zhao: Tel, 86-21-62933765; E-mail, [email protected]Search for more papers by this author

Zixin Deng,

Corresponding Author

Zixin Deng

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

*Corresponding authors: Zinxin Deng: Tel, 86-21-62933404; E-mail, [email protected] Chunhua Zhao: Tel, 86-21-62933765; E-mail, [email protected]Search for more papers by this author

Danfeng Song,

Danfeng Song

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

Search for more papers by this author

Jane Coughlin,

Jane Coughlin

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

Search for more papers by this author

Jianhua Ju,

Jianhua Ju

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

Search for more papers by this author

Xiufen Zhou,

Xiufen Zhou

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

Search for more papers by this author

Ben Shen,

Ben Shen

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA

National Cooperative Drug Discovery Group of University of Wisconsin, University of Wisconsin, Madison, Wisconsin 53705, USA

Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705, USA

Search for more papers by this author

Chunhua Zhao,

Corresponding Author

Chunhua Zhao

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

*Corresponding authors: Zinxin Deng: Tel, 86-21-62933404; E-mail, [email protected] Chunhua Zhao: Tel, 86-21-62933765; E-mail, [email protected]Search for more papers by this author

Zixin Deng,

Corresponding Author

Zixin Deng

Laboratory of Microbial Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China

*Corresponding authors: Zinxin Deng: Tel, 86-21-62933404; E-mail, [email protected] Chunhua Zhao: Tel, 86-21-62933765; E-mail, [email protected]Search for more papers by this author

First published: 21 April 2008

https://doi.org/10.1111/j.1745-7270.2008.00408.x

Citations: 1

This study was supported by the grants from the “973 Program” of the Ministry of Science and Technology (No. 2003CB114205), the National Natural Science Foundation of China (No. 30470941), and the Shanghai Municipal Council of Science and Technology (No. 04JC14058)

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Abstract

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and non-ribosomal pep-tide enzyme. Tandem ketosynthase (KS) domains (KS_10–1 and KS_10–2) were characterized and they show significant homol-ogy with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains totest the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS_10–2 is essential for OZM biosynthesis but KS_10–1 is not indispensable and might serve as a “redundant” domain. These results confirmed the existence of an “extra domain” in complex polyketide synthase.

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Volume40, Issue4

April 2008

Pages 319-326

Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

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Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

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