Improving the production of 9 alpha-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Delta(1)-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum

doi:10.1186/s12934-023-02052-y

	Improving the production of 9 alpha-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Delta(1)-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum
	Liu, Xiangcen 1,2; Zhang, Jingxian 1,2; Yuan, Chenyang1,3 ; Du, Guilin1,3 ; Han, Suwan 1,2; Shi, Jiping1,3 ; Sun, Junsong1,3 ; Zhang, Baoguo 1
	2023-03-16
发表期刊	MICROBIAL CELL FACTORIES
EISSN	1475-2859
卷号	22 期号:1
发表状态	已发表
DOI	10.1186/s12934-023-02052-y
摘要	Background 9 alpha-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking delta(1)-dehydrogenation and regulating metabolic flux.Results Five 3-Ketosteroid-delta(1)-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MF delta kstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MF delta kstD could remarkably reduce the contents of 9,21 -dihydroxy-20-methyl-pregna-4-en-3-one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MF delta kstD delta opccr successfully eliminating the two by-products. Compared with MF delta kstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9-OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol.Conclusions KstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.
关键词	9 alpha-hydroxy-4-androstene-3 17-dione Phytosterol 3-ketosteroid- increment (1)-dehydrogenase Mycobacterium fortuitum
URL	查看原文
收录类别	SCI
语种	英语
资助项目	National Key R&D Program of China[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:000953087500001
出版者	BMC
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/289995
专题	生命科学与技术学院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_史吉平组生命科学与技术学院_博士生
通讯作者	Sun, Junsong; Zhang, Baoguo
作者单位	1.Chinese Acad Sci, Shanghai Adv Res Inst, Lab Biorefinery, 99 Haike Rd, Shanghai 201210, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 3.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China
通讯作者单位	生命科学与技术学院
推荐引用方式 GB/T 7714	Liu, Xiangcen,Zhang, Jingxian,Yuan, Chenyang,et al. Improving the production of 9 alpha-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Delta(1)-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum[J]. MICROBIAL CELL FACTORIES,2023,22(1).
APA	Liu, Xiangcen.,Zhang, Jingxian.,Yuan, Chenyang.,Du, Guilin.,Han, Suwan.,...&Zhang, Baoguo.(2023).Improving the production of 9 alpha-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Delta(1)-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum.MICROBIAL CELL FACTORIES,22(1).
MLA	Liu, Xiangcen,et al."Improving the production of 9 alpha-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Delta(1)-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum".MICROBIAL CELL FACTORIES 22.1(2023).