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

Abstract:

: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 (KS 10-1 and KS 10-2) 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 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.

authors

Song D,Coughlin J,Ju J,Zhou X,Shen B,Zhao C,Deng Z

doi

10.1111/j.1745-7270.2008.00408.x

subject

Has Abstract

pub_date

2008-04-01 00:00:00

pages

319-26

issue

4

eissn

1672-9145

issn

1745-7270

journal_volume

40

pub_type

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