Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Nobuhiko Nomura, Kwang Pil Choi, Mitsuo Yamashita, Hiroko Yamamoto, Yoshikatsu Murooka

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Streptomyces cholesterol oxidase was produced in Escherichia coli by a modification of the cholesterol oxidase gene (choA′) in which the native codons for the precursor NH2-terminal region and the ribosome binding site were substituted for those favored by E. coli. The choA′ gene was expressed under the control of the lac or tac promoter in a multiple copy plasmid vector, although no expression of the native choA gene from Streptomyces was observed in E. coli. E. coli cells carrying the plasmid, pCo117, produced 2-fold more cholesterol oxidase intracellularly during 18-h culture than did the producing strain of Streptomyces sp. SA-COO cultured for 4 d. The NH2-terminal amino acid sequence of cholesterol oxidase produced by E. coli appeared to be processed between Ala20 and Ala21 of the precursor enzyme, while the Streptomyces enzyme was processed between Ala42 and Asp43. Based on the facts that the cholesterol oxidase was stable, could be assayed rapidly, and no endogenous cholesterol oxidase activity was found in any enteric bacteria, we developed two widely applicable, new promoter-probe vectors posessing the choA′ gene, multiple cloning sites, and either a low or high copy number plasmid. Since these plasmids can replicate in enteric bacteria, the new plasmid vectors have a great potential for use in enteric bacteria without the isolation of Cho- mutants.

Original languageEnglish
Pages (from-to)410-416
Number of pages7
JournalJournal of Fermentation and Bioengineering
Volume79
Issue number5
DOIs
Publication statusPublished - 1995
Externally publishedYes

Keywords

  • Streptomyces sp
  • cholesterol oxidase
  • enteric bacteria
  • promoter-probe vector

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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