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Applied Biochemistry and Biotechnology

, Volume 187, Issue 4, pp 1238–1254 | Cite as

Enhancing Expression of 3-Ketosteroid-9α-Hydroxylase Oxygenase, an Enzyme with Broad Substrate Range and High Hydroxylation Ability, in Mycobacterium sp. LY-1

  • Hui Li
  • Xiangdong Wang
  • Longfei Zhou
  • Yang Ma
  • Wanjuan Yuan
  • Xiaomei Zhang
  • Jinsong ShiEmail author
  • Zhenghong Xu
Article

Abstract

3-Ketosteroid-9α-hydroxylase (KSH) consists of two protein systems, KshA and KshB, and is a key enzyme in microbial degradation pathway of natural sterols. 9α-Hydroxy-4-androstene-3,17-dione (9α-OH-AD) is a valuable steroid pharmaceutical intermediate. The expression of a 3-ketosteroid-9α-hydroxylase oxygenase (KshA1) with a broad substrate range and high hydroxylation ability was enhanced in Mycobacterium sp. LY-1 to improve the yield of 9α-OH-AD. Through whole-genome sequence mining and homologous comparison, the putative genes (kshA1 and kshB) in wild strain LY-1 were firstly identified. Then they were heterogeneously co-expressed in Escherichia coli BL21. The transformation results of recombinant BL21-KshA1/B demonstrated KshA1/B had high hydroxylation ability to AD. Moreover, substrate preference analysis suggested that KshA1LY-1 had a broad substrate range. After enhancing expression of kshA1 and kshB in the strain LY-1, the maximum productivity of 9α-OH-AD in recombinant LY-1-KshA1/B reached 0.064 g/L/h in a 5-L stirred fermenter.

Keywords

Mycobacterium sp. LY-1 3-Ketosteroid-9α-hydroxylase oxygenase Hydroxylation 9α-OH-AD 

Notes

Funding Information

This research was financially supported by the National 863 High Tech Program of China (No. 2011AA02A211), Postgraduate Research & Practice Innovation Program of Jiangsu Provence (SJCX17_0502), and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015B146).

Compliance with Ethical Standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interest

The authors declare that there is no conflict of interest.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hui Li
    • 1
  • Xiangdong Wang
    • 1
  • Longfei Zhou
    • 1
  • Yang Ma
    • 1
  • Wanjuan Yuan
    • 1
  • Xiaomei Zhang
    • 1
  • Jinsong Shi
    • 1
    Email author
  • Zhenghong Xu
    • 1
    • 2
  1. 1.School of Pharmaceutical ScienceJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.National Engineering Laboratory for Cereal Fermentation Technology, School of BiotechnologyJiangnan UniversityWuxiPeople’s Republic of China

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