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Alternative method for site-directed mutagenesis of complex polyketide synthase in streptomyces albus ja3453

Academic Article
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Overview

authors

  • Song, D. F.
  • Coughlin, J.
  • Ju, J. H.
  • Zhou, X. F.
  • Shen, Ben
  • Zhao, C. H.
  • Deng, Z. X.

publication date

  • April 2008

journal

  • Acta Biochimica et Biophysica Sinica  Journal

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.

subject areas

  • Enzyme Activation
  • Enzyme Stability
  • Mutagenesis, Site-Directed
  • Polyketide Synthases
  • Protein Engineering
  • Streptomyces
  • Structure-Activity Relationship
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Research

keywords

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

International Standard Serial Number (ISSN)

  • 1672-9145

Digital Object Identifier (DOI)

  • 10.1111/j.1745-7270.2008.00408.x

PubMed ID

  • 18401530
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Additional Document Info

start page

  • 319

end page

  • 326

volume

  • 40

issue

  • 4

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