Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01
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- 作者：Wei Wei (1)
Shu-Yue Fan (1)
Feng-Qing Wang (1)
Dong-Zhi Wei (1) - 关键词：1 ; 4 ; Androstadiene ; 3 ; 17 ; dione ; 3 ; Ketosteroid Δ1 ; dehydrogenase ; 4 ; Androstene ; 3 ; 17 ; dione ; Gene overexpression ; Phytosterols ; Plasmid stability
- 刊名：World Journal of Microbiology and Biotechnology
- 出版年：2014
- 出版时间：July 2014
- 年：2014
- 卷：30
- 期：7
- 页码：1947-1954
- 全文大小：
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Shu-Yue Fan (1)
Feng-Qing Wang (1)
Dong-Zhi Wei (1)
1. Newworld Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China - ISSN：1573-0972
文摘
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.