Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae

IF 3.3 2区生物学 Q2 CHEMISTRY, MEDICINAL

Journal of Natural Products Pub Date : 2024-01-26 DOI:10.1021/acs.jnatprod.3c00674

Yun Lu, Yuan Liu, Yifeng Zhang, Haiyun Gao, Xiaochao Chen, Lichan Tu, Yunfeng Luo, Zhouqian Jiang, Yan Yin, Jiawei Zhou, Tianyuan Hu, Xiaoyi Wu, Jiadian Wang*, Wei Gao* and Luqi Huang*,

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引用次数: 0

Abstract

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L^–1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

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Celastrol 生物合成过程中细胞色素 P450 CYP716C52 的特征及其在工程酿酒酵母中的应用。

天南星烯醇是一种具有生物活性的五环三萜类化合物，主要分布于天南星科植物中，具有良好的治疗效果。虽然有报道称一些酶参与了天南星醇的生物合成途径，但许多生物合成步骤仍然未知。在此，研究人员整合了天南星科多个物种的转录组学和代谢图谱，以筛选与芹菜醇生物合成密切相关的细胞色素 P450（CYPs）。研究发现，CYP716酶TwCYP716C52能够氧化青芹醇的前体多春烯酸的C-2位，形成白黎芦醇酸C；在Tripterygium wilfordii悬浮细胞中通过RNAi介导抑制TwCYP716C52，进一步证实了它参与了青芹醇的生物合成。通过分子对接和定点突变实验，进一步探索了 TwCYP716C52 的 C-2 催化机制。此外，研究人员还采用模块优化策略构建了一种工程酵母菌，其生产的白藜芦醇 C 的滴度为 5.8 mg-L-1。这项研究阐明了青霉烯醇的生物合成途径，并为进一步发现酶提供了重要的功能基因和充足的前体。

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

Journal of Natural Products 生物-药学

CiteScore

9.10

自引率

5.90%

发文量

294

审稿时长

2.3 months

期刊介绍： The Journal of Natural Products invites and publishes papers that make substantial and scholarly contributions to the area of natural products research. Contributions may relate to the chemistry and/or biochemistry of naturally occurring compounds or the biology of living systems from which they are obtained. Specifically, there may be articles that describe secondary metabolites of microorganisms, including antibiotics and mycotoxins; physiologically active compounds from terrestrial and marine plants and animals; biochemical studies, including biosynthesis and microbiological transformations; fermentation and plant tissue culture; the isolation, structure elucidation, and chemical synthesis of novel compounds from nature; and the pharmacology of compounds of natural origin. When new compounds are reported, manuscripts describing their biological activity are much preferred. Specifically, there may be articles that describe secondary metabolites of microorganisms, including antibiotics and mycotoxins; physiologically active compounds from terrestrial and marine plants and animals; biochemical studies, including biosynthesis and microbiological transformations; fermentation and plant tissue culture; the isolation, structure elucidation, and chemical synthesis of novel compounds from nature; and the pharmacology of compounds of natural origin.