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

作者：Song, Danfeng; Coughlin, Jane; Ju, Jianhua; Zhou, Xiufen; Shen, Ben; Zhao, Chunhua^*; Deng, Zixin

来源：Acta Biochimica et Biophysica Sinica, 2008, 40(4): 319-326.

DOI：10.1111/j.1745-7270.2008.00408.x

• oxazolomycin
• polyketide synthase
• RecA-mediated homologous recombination
• Streptomyces
• tandem
• ketosynthase

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and non-ribosomal peptide enzyme. Tandem ketosynthase (KS) domains (KS10-1 and KS10-2) were characterized and they show significant homology 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 to test the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS10-2 is essential for OZM biosynthesis but KS10-1 is not indispensable and might serve as a "redundant" domain. These results confirmed the existence of an "extra domain" in complex polyketide synthase.

• 出版日期2008-4
• 单位上海交通大学