Engineering Regioselectivity of P450 BM3 Enables the Biosynthesis of Murideoxycholic Acid by 6β-Hydroxylation of Lithocholic Acid

IF 3.2 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS Biotechnology Journal Pub Date : 2024-11-08 DOI:10.1002/biot.202400518

Fangzhi Deng, Zhenru Zhou, Zhen Du, Mohamed Mohany, Qunyue Wu, Weiyang Liang, Lei Zhang, Shan Li

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Abstract

Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.

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对 P450 BM3 的区域选择性进行工程改造，通过对石胆酸进行 6β 羟基化来实现 Murideoxycholic Acid 的生物合成。

睾丸脱氧胆酸（Murideoxycholic acid，MDCA）是啮齿动物体内α/β-睾丸胆酸代谢产生的一种重要的次级胆汁酸，是维持胆汁酸平衡的重要成分。然而，MDCA 的生物合成仍然是一项具有挑战性的任务。在这里，我们采用半理性蛋白质工程技术，从巨型芽孢杆菌（Bacillus megaterium）中培育出了细胞色素 P450 单加氧酶 CYP102A1（P450 BM3）。经过三轮诱变，发现了一个三重变体（T260G/G328A/L82V），它能熟练催化石胆酸（LCA）的 6β- 羟基化，从而生成 MDCA，与模板 P450 BM3 突变体相比，产量提高了 8.5 倍，令人印象深刻。MDCA 的选择性也从 62.0% 提高到 96.3%。这种生物催化剂为从 LCA 生物合成 MDCA 引入了一种新方法。此外，还利用分子对接和动力学模拟揭示了 LCA 转化率和 MDCA 选择性提高的分子机制。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine

CiteScore

8.90

自引率

2.10%

发文量

123

审稿时长

1.5 months

期刊介绍： Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.