Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker.
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.
van der Geize
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Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Goningen, The Netherlands.
Hessels
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GI
van Gerwen
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R
van der Meijden
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Dijkhuizen
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L
D017353:Gene Deletion; D017930:Genes, Reporter; D005819:Genetic Markers; D006602:Hexosyltransferases / Q000235:genetics*; D008956:Models, Chemical; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics*
D005819:Genetic Markers; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase; D006602:Hexosyltransferases; C021513:levansucrase