Journal
FEMS MICROBIOLOGY LETTERS
Volume 205, Issue 2, Pages 197-202Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0378-1097(01)00464-5
Keywords
unmarked gene deletion; 3-ketosteroid dehydrogenase; isoenzyme; steroid bioconversion; Rhodococcus
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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 Delta (I)-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sac B 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 9 alpha -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 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. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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