南沙链霉菌来源细胞色素P450酶CYP154C34的表征及生物转化

Characterization and biotransformation of cytochrome P450 CYP154C34 from Streptomyces nanshensis

摘要:

【目的】P450酶作为一种多功能生物催化剂，可在温和条件下高区域和立体选择性地催化复杂化合物中未活化的C-H键，因此P450酶在化工原料合成、环境污染物降解及药物合成等领域都具有重要作用。本文对南沙链霉菌基因组中的一个新颖的P450酶CYP154C34进行研究，通过构建异源表达和全细胞生物转化重组菌探究其功能。【方法】构建2种全细胞生物转化BL21（DE3）重组菌（含pET28a-CYP154C34-RhFRED和pET28a-CYP154C34+pACYCDuet-Pdx/PdR）和1种异源表达BL21（DE3）重组菌（含pET28a-CYP154C34）。通过全细胞生物转化的方式筛选底物，分析催化功能及产物结构。比较2种全细胞生物转化重组菌和体外酶反应对底物的转化率。分析CYP154C34和不同底物及底物类似物的亲和力。【结果】通过底物筛选和产物鉴定发现CYP154C34可催化包括孕酮、睾酮、雄烯二酮在内的9种甾体化合物16α位羟基化。通过2种不同还原伴侣的全细胞体系及体外酶反应对底物转化率的比较，发现含有pET28a-CYP154C34-RhFRED的BL21（DE3）重组菌的转化率最高，可对孕酮、睾酮、雄烯二酮等7种底物实现超过90%的转化率。通过亲和力分析发现CYP154C34对底物化合物的亲和力比类似物高。【结论】本研究构建了2种CYP154C34的全细胞生物转化重组菌并鉴定其功能。CYP154C34催化的底物谱宽并且具有极高的区域和立体选择性及较高的转化率，具有良好的工业应用前景。

Abstract:

[Objective] Cytochrome P450 can catalyze the oxidation of unactivated C-H bonds in complex compounds with high regio- and stereoselectivity under mild conditions. They are multifunctional biocatalysts which play important roles in the synthesis of chemical raw materials, the degradation of environmental pollutants, and drug synthesis. This paper probed into the functions of a novel cytochrome P450 enzyme named CYP154C34 from Streptomyces nanshensis by heterologous expression and whole-cell biotransformation. [Methods] We constructed two recombinant BL21(DE3) strains for whole-cell biotransformation harboring pET28a-CYP154C34-RhFRED and pET28a-CYP154C34+pACYCDuet-Pdx/PdR, respectively. Additionally, we generated a recombinant BL21(DE3) strain for heterologous expression containing pET28a-CYP154C34. We employed whole-cell biotransformation to screen the substrates and analyzed product structures using standard methods. We then compared the substrate conversion rates of the two strains of whole-cell biotransformation with those of enzyme reactions. Finally, we analyzed the substrate affinity and analogues of CYP154C34.[Results] Nine steroids hydroxylated at the 16α position by CYP154C34 were identified, including progesterone, testosterone, and androstenedione. The BL21(DE3) strain carrying pET28a-CYP154C34-RhFRED showed the highest substrate conversion rate, achieving over 90% conversion rates for seven substrates including progesterone, testosterone, and androstenedione. In addition, CYP154C34 had stronger affinity to its substrates than the analogues. [Conclusion] This study constructed two recombinant strains of CYP154C34 for whole-cell biotransformation and identified their catalytic functions. CYP154C34 can efficiently hydroxylate a wide range of steroid substrates at the 16α position with high conversion rates and excellent regio- and stereoselectivity, serving as a promising candidate for industrial applications.