Enhancing Expression of 3-Ketosteroid-9α-Hydroxylase Oxygenase, an Enzyme with Broad Substrate Range and High Hydroxylation Ability, in Mycobacterium sp. LY-1
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.
KeywordsMycobacterium sp. LY-1 3-Ketosteroid-9α-hydroxylase oxygenase Hydroxylation 9α-OH-AD
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.
- 4.Marsheck, W. J., Kraychy, S., & Muir, R. D. (1972). Microbial degradation of sterols. Applied Microbiology 23(1), 72–77.Google Scholar
- 5.Zhang, W., Shao, M., Rao, Z., Xu, M., Zhang, X., Yang, T., Li, H., & Xu, Z. (2013). Bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum JC-12. Journal of Steroid Biochemistry and Molecular Biology, 135, 36–42.CrossRefGoogle Scholar
- 11.Ahmad, S., Roy, P. K., Basu, S. K., & Johri, B. N. (1993). Cholesterol side-chain cleavage by immobilized cells of Rhodococcus equi DSM 89-133. Indian Journal of Experimental Biology, 31, 319–322.Google Scholar
- 12.Jekkel Nee, B. A., Albrecht, K., Ambrus, G., Lang, T., Szabo, I. M., Ilkoy, E., Konczol, K., Moravcsik, I., Hantos, G., & Simonovits, E. (1991). Microbiological process for preparing 9α-hydroxy-4-androstene-3,17-dione. US.Google Scholar
- 16.Yuan, J., Chen, G., Cheng, S., Ge, F., Qiong, W., Li, W., & Li, J. (2015). Accumulation of 9α-hydroxy-4-androstene-3,17-dione by co-expressing kshA and kshB encoding component of 3-ketosteroid-9α-hydroxylase in Mycobacterium sp. NRRL B-3805. Chinese Journal of Biotechnology, 31, 523–533.Google Scholar
- 18.Capyk, J. K., Casabon, I., Gruninger, R., Strynadka, N. C., & Eltis, L. D. (2011). Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis. Journal of Biological Chemistry, 286(47), 40717–40724.CrossRefGoogle Scholar
- 20.Geize, R. V. D., Hessels, G. I., Gerwen, R. V., Meijden, P. V. D., & Dijkhuizen, L. (2002). Molecular and functional characterization of kshA and kshB , encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Molecular Microbiology, 45, 1007–1018.CrossRefGoogle Scholar
- 23.Geize, R. V. D., Hessels, G. I., Gerwen, R. V., Meijden, P. V. D., & Dijkhuizen, L. (2001). Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ1-dehydrogenase in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker. FEMS Microbiology Letters, 205(2), 197–202.CrossRefGoogle Scholar
- 24.Andor, A., Jekkel, A., Hopwood, D. A., Jeanplong, F., Ilky, V., Knya, A., Kurucz, I., & Ambrus, G. (2006). Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9α-hydroxylase in Mycobacterium smegmatis mc2 155. Applied and Environmental Microbiology, 72(10), 6554–6559.CrossRefGoogle Scholar
- 26.Fan, S., Wei, W., Wang, F., & Wei, D. (2009). Cloning, heterologous expression and purification of a 3-ketosteroid-9α-hydroxylase (KSH) from Mycobacterium sp. NwIB-01. Chinese Journal of Biotechnology, 25, 2014–2021.Google Scholar
- 28.Ma, Y., Wang, X., Wang, M., Li, H., Shi, J., & Xu, Z. (2017). Mutation breeding of high 9α-hydroxy-androst-4-ene-3,17-dione transforming strains from phytosterols and their conversion process optimization. Chinese Journal of Biotechnology, 33, 1198–1206.Google Scholar
- 33.Yao, K., Xu, L., Wang, F., & Wei, D. (2014). Characterization and engineering of 3-ketosteroid-Δ1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols. Metabolic Engineering, 24, 181–191.CrossRefGoogle Scholar
- 35.Yao, K. (2014). Investigation into the molecular mechanism of microbial sterol degradation and its metabolic engineering for the production of steroid pharmaceutical precursors. PhD Thesis, East China University of Science and Technology, Shanghai, China.Google Scholar
- 37.Wang, G. E., Chen, Y. Q., Fan, Y. X., Li, J. F., & Chen, X. L. (2016). Enhancement expression of 3-phytosterone-9α-hydroxylase in Mycobacterium. Pharmaceutical Biotechnology, 23, 381–384.Google Scholar