title: Improvement of NADPH-dependent P450-mediated biotransformation of 7α,15α-diOH-DHEA from DHEA by a dual cosubstrate-coupled system.
abstract: Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini. By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini. Using 20mM nicotinic acid and 15g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21g/L could be achieved after 60h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation.
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Xiao-Mei
authors.initials: XM
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Jin-Song
authors.initials: JS
authors.lastname: Gong
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Heng
authors.initials: H
authors.lastname: Li
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Rao
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Jin-Song
authors.initials: JS
authors.lastname: Shi
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People's Republic of China. Electronic address: zhenghxu@jiangnan.edu.cn.
authors.forename: Zheng-Hong
authors.initials: ZH
authors.lastname: Xu
mesh_terms: D001711:Biotransformation; D003067:Coenzymes / Q000378:metabolism; D020231:Colletotrichum / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000378:metabolism*; D006900:Hydroxylation; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D003067:Coenzymes; D003687:Dehydroepiandrosterone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System
keywords: Biotransformation; Colletotrichum lini; DHEA; NADPH