Engineering of redox partners and cofactor NADPH supply of CYP68JX for efficient steroid two-step ordered selective hydroxylation activity.

Autor: Liu W; School of Biotechnology, Jiangnan University, Wuxi 214122, China., Li H; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China. Electronic address: lihui@jiangnan.edu.cn., Guo D; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Ni Y; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Zhang X; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Shi J; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Koffas MAG; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, United States. Electronic address: koffam@rpi.edu., Xu Z; National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China; Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi 214122, China.
Jazyk: angličtina
Zdroj: The Journal of steroid biochemistry and molecular biology [J Steroid Biochem Mol Biol] 2024 Apr; Vol. 238, pp. 106452. Date of Electronic Publication: 2023 Dec 29.
DOI: 10.1016/j.jsbmb.2023.106452
Abstrakt: CYP68JX, a P450 hydroxylase, derived from Colletotrichum lini ST-1 is capable of biotransforming dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA). Redox partners and cofactor supply are important factors affecting the catalytic activity of CYP68JX. In this study, the heterologous expression of CYP68JX in Saccharomyces cerevisiae BY4741 was realized resulting in a 17.1% target product yield. In order to increase the catalytic efficiency of CYP68JX in S. cerevisiae BY4741, a complete cytochrome P450 redox system was constructed. Through the combination of CYP68JX and heterologous CPRs, the yield of the target product 7α,15α-diOH-DHEA in CYP68JX recombinant system was increased to 37.8%. Furthermore, by adding NADPH coenzyme precursor tryptophan of 40 mmol/L and co-substrate fructose of 20 g/L during the conversion process, the catalytic efficiency of CYP68JX was further improved, the target product yield reached 57.9% which was 3.39-fold higher than initial yield. Overall, this study provides a reference for improving the catalytic activity of P450s.
(Copyright © 2024 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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