Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker

FEMS Microbiol Lett. 2001 Dec 18;205(2):197-202. doi: 10.1016/s0378-1097(01)00464-5.


This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Delta1-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain SQ1 was used to avoid its random genomic integration. The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD). Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R. erythropolis SQ1. UV mutants of strain RG1 unable to grow on AD were isolated. One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD. It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g x l(-1). The two KSTD enzymes apparently both function in AD and 9OHAD catabolism. These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Deletion
  • Genes, Reporter
  • Genetic Markers
  • Hexosyltransferases / genetics*
  • Models, Chemical
  • Mutation
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Rhodococcus / enzymology
  • Rhodococcus / genetics*


  • Genetic Markers
  • Oxidoreductases
  • 3-oxosteroid delta(1) dehydrogenase
  • Hexosyltransferases
  • levansucrase