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Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01

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Abstract

Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ1-dehydrogenase genes (kstD M and kstD A) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD M and kstD A were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD M/kstD A overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60 %, respectively. This study on the manipulation of the main kstDM gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.

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Acknowledgments

We thank Dr. W.R. Jacobs Jr. (Howard Hughes Medical Institute, USA) for providing plasmid pMV261 and pMV306. This research was financially supported by the Fundamental Research Funds for the Central Universities of China (No. WF1114017), the National Natural Science Foundation of China (No. C050203-31200596), the School Research Funds of ECAST (No. YF0157129), the National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204), and the National Basic Research Program of China (No. 2009CB724703).

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  1. Newworld Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Wei Wei, Shu-Yue Fan, Feng-Qing Wang & Dong-Zhi Wei

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  1. Wei Wei
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  2. Shu-Yue Fan
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  3. Feng-Qing Wang
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  4. Dong-Zhi Wei
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Correspondence to Feng-Qing Wang or Dong-Zhi Wei.

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Wei Wei and Shu-Yue Fan provide the equal work in this study.

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Wei, W., Fan, SY., Wang, FQ. et al. Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01. World J Microbiol Biotechnol 30, 1947–1954 (2014). https://doi.org/10.1007/s11274-014-1614-3

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