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<p>Steroidal C12<i>β</i>/15<i>α</i>-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 C12<i>β</i>- and 99.6% for C15<i>α</i>-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 <i>Pichia pastoris</i> yields titers of 4.6 g/L 12<i>β</i>-OH progesterone, 10.9 g/L 15<i>α</i>-OH progesterone and 14.1 g/L 15<i>α</i>-OH androstenedione. These products serve as key intermediates for streamlined synthesis of C12-/C15-functionalized steroids such as drospirenone and C-<i>nor</i>-D-<i>homo</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月28日

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  • Ling Wang,
  • Ying Wang,
  • Xiaogang Peng,
  • Chenghua Gao,
  • Xinfang Wei,
  • Peng Chen,
  • Pan Xiao,
  • Qian Li,
  • Aitao Li

摘要

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.