通过大肠杆菌表达的人CYP7A1高效全细胞生物催化生产7α-羟基-4-胆固醇-3- 1。

IF 2.5 2区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Steroid Biochemistry and Molecular Biology Pub Date : 2025-09-18 DOI:10.1016/j.jsbmb.2025.106866

Qiannan Shang , Jiahui Huang , Shi Qin , Haichao Zhu , Riling Chen , Lina Yin , Qingzhong Hu

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引用次数: 0

摘要

人细胞色素P450酶CYP7A1在胆汁酸生物合成中催化限速的胆固醇7α-羟基化，但在微生物宿主中难以功能表达。在这里，我们报道了一个全细胞大肠杆菌生物催化平台，使用双电子或双启动子载体系统共表达截断的CYP7A1及其氧化还原伙伴细胞色素P450还原酶（CPR）。通过GroES-GroEL共表达提高蛋白表达，最终确定Rosetta(DE3)-pET-tCYP7A1-tCPR为最佳菌株。在羟丙基-β-环糊精和多粘菌素B的帮助下，7α-羟基-4-胆甾醇-3- 1 （7α-HCO）以118.3mg·L⁻¹·d⁻，纯度为bb0 98%。产物经核磁共振和高分辨率质谱分析验证。这项工作提出了第一个通过人类CYP7A1进行7α-HCO生物合成的微生物系统，为类固醇生产和药物代谢和抑制剂筛选提供了可扩展的策略。

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Efficient whole-cell biocatalytic production of 7α-hydroxy-4-cholesten-3-one via human CYP7A1 expressed in Escherichia coli

Human cytochrome P450 enzyme CYP7A1 catalyzes the rate-limiting 7α-hydroxylation of cholesterol in bile acid biosynthesis but is difficult to express functionally in microbial hosts. Here, we report a whole-cell Escherichia coli biocatalytic platform co-expressing truncated CYP7A1 and its redox partner cytochrome P450 reductase (CPR) using bicistronic or dual-promoter vector systems. Protein expression was improved via GroES-GroEL co-expression, and Rosetta(DE3)-pET-tCYP7A1-tCPR was identified as the optimal strain. With the aid of hydroxypropyl-β-cyclodextrin and polymyxin B, 7α-hydroxy-4-cholesten-3-one (7α-HCO) was produced at 118.3 mg·L⁻¹ ·d⁻¹ with > 98 % purity. The product was verified by NMR and high-resolution mass spectrometry. This work presents the first microbial system for 7α-HCO biosynthesis via human CYP7A1, offering a scalable strategy for steroid production and potential applications in drug metabolism and inhibitor screening.

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来源期刊

Journal of Steroid Biochemistry and Molecular Biology 生物-内分泌学与代谢

CiteScore

8.60

自引率

2.40%

发文量

113

审稿时长

46 days

期刊介绍： The Journal of Steroid Biochemistry and Molecular Biology is devoted to new experimental and theoretical developments in areas related to steroids including vitamin D, lipids and their metabolomics. The Journal publishes a variety of contributions, including original articles, general and focused reviews, and rapid communications (brief articles of particular interest and clear novelty). Selected cutting-edge topics will be addressed in Special Issues managed by Guest Editors. Special Issues will contain both commissioned reviews and original research papers to provide comprehensive coverage of specific topics, and all submissions will undergo rigorous peer-review prior to publication.