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Applied and Environmental Microbiology, May 2000, p. 2029-2036, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Delta 1-Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

R. van der Geize,1 G. I. Hessels,1 R. van Gerwen,2 J. W. Vrijbloed,1,dagger P. van der Meijden,2 and L. Dijkhuizen1,*

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9750 AA Haren,1 and Diosynth bv, AkzoNobel, 5340 BH Oss,2 The Netherlands

Received 28 December 1999/Accepted 25 February 2000

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta 1-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta 1-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta 1-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha -hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta 1-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta 1-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

* Corresponding author. Mailing address: L. Dijkhuizen, Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands. Phone: 31 (50) 3632153. Fax: 31 (50) 3632154. E-mail: L.Dijkhuizen{at}biol.rug.nl.

dagger Present address: Organic-Chemistry Institute, University Zürich, CH-8057 Zürich, Switzerland.

Applied and Environmental Microbiology, May 2000, p. 2029-2036, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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