Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

doi:10.1186/s12934-021-01717-w

	Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment
	Yuan, Chen-Yang1,2,3 ; Ma, Zhi-Guo 1; Zhang, Jing-Xian 1,3; Liu, Xiang-Cen 1,3; Du, Gui-Lin1,2,3 ; Sun, Jun-Song1,2,3 ; Shi, Ji-Ping1,2,3 ; Zhang, Bao-Guo 1,3
	2021-12-23
发表期刊	MICROBIAL CELL FACTORIES (IF:4.3[JCR-2023],5.5[5-Year])
EISSN	1475-2859
卷号	20 期号:1
发表状态	已发表
DOI	10.1186/s12934-021-01717-w
摘要	Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Delta(1)-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Delta(1)-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a beta-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Delta(1)-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Delta(1)-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.
关键词	9 21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) kstD hsd4A fadA5 Intracellular environment
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收录类别	SCI ; SCIE
语种	英语
资助项目	National Key R&D Program of Chin[2017YFE0112700] ; Natural Science Foundation of China[21906139]
WOS研究方向	Biotechnology & Applied Microbiology
WOS类目	Biotechnology & Applied Microbiology
WOS记录号	WOS:000733738400001
出版者	BMC
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/145753
专题	生命科学与技术学院_博士生生命科学与技术学院_特聘教授组_孙俊松组生命科学与技术学院_特聘教授组_史吉平组
通讯作者	Sun, Jun-Song; Zhang, Bao-Guo
作者单位	1.Chinese Acad Sci, Shanghai Adv Res Inst, Lab Biorefinery, 99 Haike Rd, Shanghai 201210, Peoples R China 2.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
第一作者单位	生命科学与技术学院
通讯作者单位	生命科学与技术学院
推荐引用方式 GB/T 7714	Yuan, Chen-Yang,Ma, Zhi-Guo,Zhang, Jing-Xian,et al. Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment[J]. MICROBIAL CELL FACTORIES,2021,20(1).
APA	Yuan, Chen-Yang.,Ma, Zhi-Guo.,Zhang, Jing-Xian.,Liu, Xiang-Cen.,Du, Gui-Lin.,...&Zhang, Bao-Guo.(2021).Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.MICROBIAL CELL FACTORIES,20(1).
MLA	Yuan, Chen-Yang,et al."Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment".MICROBIAL CELL FACTORIES 20.1(2021).