Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3,ACS Catalysis

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Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3
ACS Catalysis ( IF 11.3 ) Pub Date : 2024-03-04 , DOI: 10.1021/acscatal.3c06137
Qihang Chen _{1,

2} , Zikai Chao _{1,

2} , Ke Wang _{1,

2} , Xinglong Wang _{1,

2} , Hao Meng ₃ , Xirong Liu ₃ , Xiaoyu Shan _{1,

2} , Jingwen Zhou _{1,

2,

4}

Affiliation

Synthesis of corticosteroids, particularly hydrocortisone, is challenging owing to the complex network requiring pairing of cytochrome P450s with cytochrome P450 reductase (CPR) for achieving regionally selective hydroxylation modifications at multiple sites. Herein, we engineered a self-sufficient P450BM3 (CYP102A1 from Bacillus megaterium) for effectively reducing the traditionally complex, multienzyme cascade process (three steps and six enzymes) of hydrocortisone synthesis from progesterone (PG) to a simplified two-step process involving at least two enzymes. Driven by computational simulation-guided substrate access channel and heme center pocket engineering, a series of P450BM3 variants were gradually designed with the ability to catalyze C16β, C17α, C21, and C17α/21 oxidation of PG and C11α oxidation of cortexolone (c). Subsequently, molecular dynamics simulations with an oxy-ferrous model of P450BM3 variants revealed that the glycine mutations of residues that are repulsive to the substrate allow for more stable exposure of the substrate above Fe═O. Finally, the developed P450 variants were employed to construct efficient Escherichia coli catalytic systems, which further achieved 11α/β-hydrocortisone (f/e) production in one pot from 1 g/L PG at a molar conversion rate of 81 and 84% (912 and 955 mg/L), respectively. Thus, this study provides feasible strategies for simplifying the biosynthetic steps and biocatalysts for steroidal pharmaceutical production.

中文翻译：

通过工程化 P450BM3 简化皮质类固醇生物合成途径

皮质类固醇，特别是氢化可的松的合成具有挑战性，因为复杂的网络需要细胞色素 P450 与细胞色素 P450 还原酶 (CPR) 配对，以实现多个位点的区域选择性羟基化修饰。在此，我们设计了一种自给自足的 P450BM3（来自巨大芽孢杆菌的 CYP102A1），用于有效地将黄体酮（PG）合成氢化可的松的传统复杂的多酶级联过程（三个步骤和六种酶）减少到简化的两步过程，至少涉及两种酶。在计算模拟引导的底物进入通道和血红素中心袋工程的驱动下，逐渐设计了一系列P450BM3变体，具有催化PG的C16β、C17α、C21和C17α/21氧化以及皮质醇的C11α氧化的能力(c )。随后，使用 P450BM3 变体的含氧亚铁模型进行分子动力学模拟表明，对底物具有排斥性的残基的甘氨酸突变使得底物能够更稳定地暴露在 Fe=O 之上。最后，开发的P450变体被用于构建高效的大肠杆菌催化系统，进一步实现了从1 g/L PG一锅生产11α/β-氢化可的松（f / e ），摩尔转化率为81%和84%（分别为 912 和 955 毫克/升）。因此，本研究为简化甾体药物生产的生物合成步骤和生物催化剂提供了可行的策略。

更新日期：2024-03-04

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