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<p><i>ent</i>-Kaurane diterpenoids (<i>ent</i>-KTs) represent a structurally diverse class of natural products renowned for their antitumor and anti-inflammatory bioactivities. The C-14 hydroxyl modification is crucial for enhancing their potency; however, introducing this functional group remains a considerable challenge. Here, we present a computational heme-guided site-specific (CHS) strategy to identify three bacterial P450s (CYP260A1, CYP105N1, and CYP154C5) for C-14 hydroxylation. Further computationally guided enzyme engineering and redox partner screening identify the CYP260A1 L162V variant paired with CamA/CamB, achieving a 52-fold increase in production titer and a yield of 84.2 mg/L of (14<i>R</i>,16<i>R</i>)-<i>ent</i>-kauran-14,16-diol (<b>2</b>) in <i>Escherichia coli</i>. Substrate scope test reveals functional groups affecting reactivity. Structure-activity relationship studies demonstrate the synergistic effect between the C-14 hydroxyl and C15–C16 Michael acceptor, resulting in a potent derivative (<b>27</b>) with strong cytotoxicity (IC<sub>50</sub><sup>HCT116</sup> = 1.4 μM). This study demonstrates a framework combining CHS-guided P450 discovery and computational enzyme engineering to advance <i>ent</i>-KT modifications.</p>

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Discovery and engineering of bacterial P450s for C-14 hydroxylation in ent-kaurane diterpenoids

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月12日

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  • Xiaoxu Lin,
  • Zhixi Xiao,
  • Xingwang Xu,
  • Xiaowei Zhang,
  • Xingming Pan,
  • Chenxi Zhu,
  • Chenxi He,
  • Li Feng,
  • Fang-Ru Li,
  • Hui-Min Xu,
  • Zhe Wang,
  • Ninghua Tan,
  • Liao-Bin Dong

摘要

ent-Kaurane diterpenoids (ent-KTs) represent a structurally diverse class of natural products renowned for their antitumor and anti-inflammatory bioactivities. The C-14 hydroxyl modification is crucial for enhancing their potency; however, introducing this functional group remains a considerable challenge. Here, we present a computational heme-guided site-specific (CHS) strategy to identify three bacterial P450s (CYP260A1, CYP105N1, and CYP154C5) for C-14 hydroxylation. Further computationally guided enzyme engineering and redox partner screening identify the CYP260A1 L162V variant paired with CamA/CamB, achieving a 52-fold increase in production titer and a yield of 84.2 mg/L of (14R,16R)-ent-kauran-14,16-diol (2) in Escherichia coli. Substrate scope test reveals functional groups affecting reactivity. Structure-activity relationship studies demonstrate the synergistic effect between the C-14 hydroxyl and C15–C16 Michael acceptor, resulting in a potent derivative (27) with strong cytotoxicity (IC50HCT116 = 1.4 μM). This study demonstrates a framework combining CHS-guided P450 discovery and computational enzyme engineering to advance ent-KT modifications.