School of Biotechnology, Jiangnan University, Wuxi 214122, China.
School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China.
J Steroid Biochem Mol Biol. 2024 Apr;238:106452. doi: 10.1016/j.jsbmb.2023.106452. Epub 2023 Dec 29.
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.
CYP68JX 是一种 P450 羟化酶，来源于 Colletotrichum lini ST-1，能够将脱氢表雄酮 (DHEA) 生物转化为 3β,7α,15α-三羟基-5-雄烯-17-酮 (7α,15α-二 OH-DHEA)。氧化还原伴侣和辅因子供应是影响 CYP68JX 催化活性的重要因素。在这项研究中，通过在酿酒酵母 BY4741 中异源表达 CYP68JX，实现了 17.1%的目标产物收率。为了提高 CYP68JX 在酿酒酵母 BY4741 中的催化效率，构建了一个完整的细胞色素 P450 氧化还原系统。通过 CYP68JX 与异源 CPRs 的结合，将目标产物 7α,15α-二 OH-DHEA 在 CYP68JX 重组系统中的产量提高到 37.8%。此外，在转化过程中添加 40mmol/L 的 NADPH 辅酶前体色氨酸和 20g/L 的共底物果糖，进一步提高了 CYP68JX 的催化效率，目标产物的产量达到 57.9%，比初始产量提高了 3.39 倍。总的来说，这项研究为提高 P450s 的催化活性提供了参考。
