Secretory overproduction of Arthrobacter simplex 3-ketosteroid Δ1-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector

K. P. Choi, Y. Murooka, I. Molnár

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

The gene for 3-ketosteroid Δ1-dehydrogenase (ksdD) of Arthrobacter simplex was expressed in Streptomyces lividans and the secreted enzyme was overproduced by using a multi-copy shuttle vector composed of pIJ702 and pUC19. Deletional analysis of the recombinant plasmid showed that the entire coding sequence of the ksdD gene was located within a 7-kb segment of the chromosomal DNA obtained from the enzyme-producing strain of A. simplex. When S. lividans carrying the recombinant plasmid was grown in an appropriate medium, the cells produced about 100-fold more 3-ketosteroid Δ1-dehydrogenase than the original strain. Although the percentage of enzyme secreted was changed during cultivation, a maximum 55% of the enzyme was secreted into the cultured medium of S. lividans, while A. simplex did not produce the enzyme extracellularly. Secretory overproduction of 3-ketosteroid Δ1-dehydrogenase in S. lividans was also identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and on native gel, and the enzyme reaction was confirmed by reverse-phase HPLC using 4-androstene-3,17-dione as a substrate.

Original languageEnglish (US)
Pages (from-to)1044-1049
Number of pages6
JournalApplied Microbiology and Biotechnology
Volume43
Issue number6
DOIs
StatePublished - Nov 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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