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

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.

图/表 10

表1 本研究所涉及的菌种信息

Tab. 1 Strains used in this study

菌株	基因型	来源
SynV	MATa his3Δ1;leu2;met15Δ;ura3-52	天津大学
SynV8	SynV: ΔGal80	本研究构建
GA0-1	SynV8: ΔHO:: FBA1p-bAS-CYC1-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-G418	本研究构建
GA0	SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3	本研究构建
GA160	SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3	本研究构建
GA180-1	SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-Gal7p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3	本研究构建

表2 5×ISO缓冲液组分

Tab. 2 Components of the 5×ISO buffer

成分	体积/μL
1 mol/L Tris-HCl（pH7.5）	3000
2 mol/L MgCl₂	150
dNTP mix	240
1 mol/L DTT	300
PEG-8000	1.5 g
0.1 mol/L NAD	300
ddH₂O	定容至6 mL，-80 ℃分装，保存

表3 Gibson反应液组分

Tab. 3 Components of Gibson reaction solution

成分	体积/μL
5×ISO buffer	320
10 U/μL T5 exonuclease	0.64
2 U/μL Phusion polymerase	20
40 U/μL	160
ddH₂O	1200

图1 跨膜域改造后CYP72A63（T338S）的TMHMM预测结果

Fig. 1 TMHMM prediction for the transmembrane-domain-modified CYP72A63 (T338S)

图2 不同跨膜结构对CYP72A63（T338S）底物选择性的影响［相同字母表明在95%的置信水平上（P<0.05）没有显著差异］

Fig. 2 Influence of the transmembrane structures on the substrate selectivity of CYP72A63 (T338S)[The same letters indicate no significant difference at the95% confidence level (P < 0.05)]

表4 不同宿主中脂质代谢相关基因的转录丰度

Tab. 4 Transcription levels of genes associated to lipid metabolism in different strains

基因

注释

FPKM

Sgib

FPKM

SynV

FAA1

long-chain fatty acid-CoA ligase FAA1

1534.17

410.28

SUR2

sphingosine C-4 hydroxylase

480.16

162.61

图3 敲除FAA1及SUR2的工程菌株产物分布［相同字母表明在95%的置信水平上（P<0.05）没有显著差异］

Fig. 3 Product profile of the strains with FAA1or SUR2 knock out[The same letters indicate no significant difference at the 95% confidence level (P < 0.05)]

图4 过表达ELO1、ELO2、ELO3工程菌中三萜化合物的产量［“*”表示在95%的置信水平上（P<0.05）存在显著差异］

Fig. 4 Production of triterpenoids in the engineered strains overexpressing ELO1, ELO2 and ELO3[Asterisk (*) indicates significant difference at the 95% confidence level (P < 0.05)]

图 5 过表达外源葡萄糖神经酰胺合酶工程菌的三萜产量［相同字母表明在95%的置信水平上（P<0.05）没有显著差异］

Fig. 5 Production of triterpenoids in the engineered strains overexpressing heterogenous GCSs[The same letters indicate no significant difference at the 95% confidence level (P < 0.05)]

图6 Uni25647表达强化后工程菌的产物的分布（a）及P450酶的相对转录丰度（b）［“*”表示在95%的置信水平上（P<0.05）存在显著差异］

Fig. 6 Product profile (a) and the relative transcription abundance of P450 enzymes after up-regulating Uni25647 expression (b)[Asterisk （*） indicates significant difference at 95% confidence level (P < 0.05)]

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