Ⅱ型细胞色素P450酶氧化β-香树脂醇的选择性调控研究

doi:10.12211/2096-8280.2021-081

合成生物学 ›› 2021, Vol. 2 ›› Issue (5): 804-814.DOI: 10.12211/2096-8280.2021-081

Ⅱ型细胞色素P450酶氧化β-香树脂醇的选择性调控研究

孙文涛¹, 张昕哲², 万盛通², 王茹雯², 李春^1,²

^1.清华大学化工系，北京 100084
^2.北京理工大学化学与化工学院，北京 100101

收稿日期:2021-08-06 修回日期:2021-09-24 出版日期:2021-10-31 发布日期:2021-11-19
通讯作者: 李春
作者简介:孙文涛（1988—），男，博士，博士后。研究方向为酶工程与合成生物学。E-mail：sunwentao@tsinghua.edu.cn|李春（1970—），男，博士生导师，教授。研究方向为生物催化与酶工程，合成生物学与代谢工程。E-mail：lichun@tsinghua.edu.cn
基金资助:
国家重点研发计划(2018YFA0901800);国家自然科学基金(21736002);清华大学“水木学者”计划(2020SM097)

Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes

Wentao SUN¹, Xinzhe ZHANG², Shengtong WAN², Ruwen WANG², Chun LI^1,²

^1.Department of Chemical Engineering，Tsinghua University，Beijing 100084，China
^2.School of Chemistry and Chemical Engineering，Beijing Institute of Technology，Beijing 100101，China

Received:2021-08-06 Revised:2021-09-24 Online:2021-10-31 Published:2021-11-19
Contact: Chun LI

摘要/Abstract

摘要：

细胞色素P450酶（简称P450酶）是天然产物合成过程中的关键修饰酶，其催化的杂泛性使得一种生物合成途径中的一个P450酶可以氧化多个中间体导致多种结构类似物的产生。P450酶催化选择性的传统调控策略主要为催化结构域的改造以及氧化还原伴侣工程，然而在由P450酶、细胞色素P450还原酶（CPR）、生物膜构成的Ⅱ型P450酶催化系统中，蛋白跨膜域、膜组分的代谢等因素对其选择性的影响尚不清晰。为探索Ⅱ型P450酶催化选择性调控的新策略，解决甘草次酸合成过程中关键Ⅱ型P450酶CYP72A63（T338S）的底物选择性差而产生副产物11-脱氧甘草次酸的问题，本文通过在CYP72A63（T338S）的N端融合酵母内源P450酶以及其他内质网定位蛋白的跨膜域方式重塑CYP72A63（T338S）的跨膜结构，改造重要膜组分鞘脂的代谢途径，调节生物合成途径中具有底物竞争关系的P450酶表达比例的三种策略，以酿酒酵母为底盘菌株，通过体内验证的方式，探究了跨膜域、鞘脂代谢以及具有底物竞争关系的P450酶表达比例的变化对CYP72A63（T338S）催化特性的影响。结果表明，跨膜域的重塑以及鞘脂代谢的调节显著改变了CYP72A63（T338S）催化的底物选择性，N端跨膜域融合NTE1N以及敲除二氢鞘氨醇4-羟化酶的编码基因SUR2显著抑制了其对β-香树脂醇的氧化选择性；过表达来自毕赤酵母的葡萄糖神经酰胺合酶则会显著促进其对β-香树脂醇的氧化选择性；通过提高Uni25647（来源于乌拉尔甘草的11-氧-β-香树脂醇合成酶）的表达比例，促进了其与CYP72A63（T338S）竞争β-香树脂醇的能力，完全抑制了副产物11-脱氧甘草次酸的合成。本文为P450酶，尤其是膜定位的Ⅱ型P450酶催化特性调控的研究提供了新的思路和方法。

关键词: 细胞色素P450酶, 跨膜域, 鞘脂, 催化选择性, 甘草次酸, 酿酒酵母

Abstract:

Cytochrome P450 enzymes are tailoring enzymes for a key role in the biosynthesis of many natural products, and their catalytic promiscuity enables diverse structures to catalyze different reactions involved in the same biosynthetic pathway, such as the oxidization of same intermediates to form undesired analogues. The traditional strategies for regulating catalytic selectivity for substrate by P450 enzymes mainly focus on engineering their catalytic domains and/or redox partners. However, roles of the transmembrane domain, metabolism of membrane components and the expression ratio of P450 enzymes catalyzing different reactions in the catalytic specificity of membrane-bound Class Ⅱ P450 enzymes have been overlooked. To explore regulation mechanism underlying the catalytic selectivity for substrate by Class Ⅱ P450 enzymes to eliminate the formation of by-product 11-deoxyglycyrrhetinic acid from the uncontrolled catalytic selectivity of CYP72A63 (T338S) during the biosynthesis of glycyrrhetinic acid, the influences of these factors were studied in vivo throughthe Saccharomyces cerevisiae expression and verification platform with three strategies: remodeling the transmembrane structure of CYP72A63 (T338S) by domain swapping with the transmembrane domain of the yeast endogenous P450 enzymes and other endoplasmic reticulum located proteins, redirecting the membrane metabolism by reconstructing the metabolic pathway of host membrane components, and regulating the expression ratio of P450 enzymes upstream and downstream the glycyrrhetinic acid synthetic pathway. Our experimental results indicate that the remodeling of the transmembrane domain and the regulation of membrane metabolism significantly changed the substrate selectivity of CYP72A63 (T338S), and the fusion of N-terminal transmembrane domain with NTE1 and the knockout of sphinganine C4-hydroxylase encoding gene SUR2 significantly inhibited the oxidation selectivity for β-amyrin by CYP72A63 (T338S), but the overexpression of glucosylceramide synthase from Pichia pastoris remarkably enhanced its oxidation selectivity for β-amyrin. Moreover, by up-regulating the expression of Uni25647, and thus enhancing its competition ability for β-amyrin against CYP72A63 (T338S), the production of 11-deoxyglycyrrhetinic acid was completely eliminated. This study provides new ideas and methods for the catalytic regulation of P450 enzymes, especially for the membrane-bound Class Ⅱ P450 enzymes.

Key words: cytochrome P450, transmembrane domain, sphingolipid, catalytic selectivity, glycyrrhetinic acid, Saccharomyces cerevisiae

中图分类号:

Q816

孙文涛, 张昕哲, 万盛通, 王茹雯, 李春. Ⅱ型细胞色素P450酶氧化β-香树脂醇的选择性调控研究[J]. 合成生物学, 2021, 2(5): 804-814.

Wentao SUN, Xinzhe ZHANG, Shengtong WAN, Ruwen WANG, Chun LI. Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes[J]. Synthetic Biology Journal, 2021, 2(5): 804-814.

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Ⅱ型细胞色素P450酶氧化β-香树脂醇的选择性调控研究

Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes

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