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[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 fromStreptomyces 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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