Divergent evolution of fungal P450 monooxygenase unlocks simultaneous access to C12β and C15α oxyfunctionalization of steroids

Ling Wang, Ying Wang, Xiaogang Peng, Chenghua Gao, Xinfang Wei, Peng Chen, Pan Xiao, Qian Li () and Aitao Li ()
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Ling Wang: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Ying Wang: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Xiaogang Peng: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Chenghua Gao: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Xinfang Wei: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Peng Chen: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Pan Xiao: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Qian Li: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences
Aitao Li: Hubei University, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: Abstract Steroidal C12β/15α-hydroxylation are pivotal in synthesizing steroid drugs but remain challenging via chemical and biological methods. To address this, structure-guided divergent evolution is applied to the fungal P450 monooxygenase CYP68J5_fg. Two optimized variants, W12M5 (F107S/Q112R/N295T/V299T/R368K) and W15M4 (Q112C/D126V/V299L/A362M) are created, achieving high selectivity (97.7% for C12β- and 99.6% for C15α-hydroxylation of progesterone) alongside enhanced catalytic efficiency, effectively overcoming the classic activity-selectivity trade-off. Molecular dynamics simulations reveal that key mutations reorient the substrate by reshaping the binding pocket’s polarity and hydrogen-bonding network, enabling hydroxylation at distinct positions. High-density fermentation with engineered Pichia pastoris yields titers of 4.6 g/L 12β-OH progesterone, 10.9 g/L 15α-OH progesterone and 14.1 g/L 15α-OH androstenedione. These products serve as key intermediates for streamlined synthesis of C12-/C15-functionalized steroids such as drospirenone and C-nor-D-homo derivatives. Collectively, this study demonstrates the successful divergent evolution of a fungal P450, a strategy which has so far not been reported in the literature, highlights its broad applicability for the scalable synthesis of complex bioactive molecules.

Date: 2025
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DOI: 10.1038/s41467-025-65736-6

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