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A mutant form of 3-ketosteroid-Δ1-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ1-dehydrogenase (KSDD), which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDDI (from M. neoaurum ST-095) and KSDDII (from M. neoaurum JC-12), we found the only difference between KSDDI and KSDDII was the mutation of Val366 to Ser366. This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDDII has a higher C1,2 dehydrogenation activity than KSDDI. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd I and ksdd II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd I and ksdd II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val366Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.

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Abbreviations

AD:

Androst-4-ene-3,17-dione

ADD:

Androst-1,4-diene-3,17-dione

KSDD:

3-Ketosteroid-Δ1-dehydrogenase

FAD:

Flavin adenine dinucleotide

Me-β-CD:

Methyl-β-cyclodextrin

PMS:

Phenazine methosulphate

DCPIP:

2,6-Dichlorophenolindophenol

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Acknowledgments

We sincerely appreciate Professor W. R. Jacobs, Jr. (Howard Hughes Medical Institute, USA) for providing plasmids pMV261 and pMV306, and Professor T. Parish (Department of Infectious & Tropical Diseases, United Kingdom) for providing plasmids of p2NIL and pGOAL19. This work was supported by the National Basic Research Program of China (973 Program) (2012CB725202), the High-Tech Research and Development Programs of China (2011AA02A211, SS2015AA021004), the National Natural Science Foundation of China (31570085), Jiangsu Province Science Fund for Distinguished Young Scholars (BK20150002), the Fundamental Research Funds for the Central Universities (JUSRP51306A), the 111 Project (111-2-06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People’s Republic of China

    Minglong Shao, Xian Zhang, Zhiming Rao, Meijuan Xu & Taowei Yang

  2. Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China

    Hui Li & Zhenghong Xu

  3. Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA

    Shangtian Yang

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  1. Minglong Shao
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Correspondence to Zhiming Rao.

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Shao, M., Zhang, X., Rao, Z. et al. A mutant form of 3-ketosteroid-Δ1-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095. J Ind Microbiol Biotechnol 43, 691–701 (2016). https://doi.org/10.1007/s10295-016-1743-9

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