胆固醇7α-羟化酶在毕赤酵母中的异源表达

doi:10.13560/j.cnki.biotech.bull.1985.2023-0387

摘要/Abstract

摘要：

胆固醇7α-羟化酶（CYP7A1）是胆固醇分解为胆汁酸的限速酶，其催化胆固醇为7α-羟基胆固醇，7α-羟基胆固醇是重要的甾体药物及中间体。以人源CYP7A1作为研究对象，首先构建了重组毕赤酵母/pPIC3.5K-CYP7A1；然后对7α-羟化酶进行分子改造，之后将酶与不同来源NADPH细胞色素氧化还原酶（CPR）适配，以提高目的产物7α-羟基胆固醇的产量。其中突变体G485A将7α-羟基胆固醇的产量提高了8.70%；5种不同来源的CPR与CYP7A1共表达毕赤酵母菌株均可提高7α-羟基胆固醇的产量，并且罗汉果（Siraitia grosvenorii）来源的CPR（SgCPR）与CYP7A1适配性最好，使7α-羟基胆固醇的产率提高了82.26%；在以上研究的基础上，构建突变体G485A与SgCPR共表达毕赤酵母菌株，最终7α-羟基胆固醇的产量提高了133.79%，其产量为0.25 mg/L。实验在毕赤酵母中成功异源表达人源胆固醇7α-羟化酶，并通过分子改造及与不同来源CPR适配的方法提高了7α-羟基胆固醇的产量。

关键词: 甾体药物, 胆固醇, 胆固醇7α-羟化酶, 7α-羟基胆固醇, 毕赤酵母

Abstract:

Cholesterol 7α-hydroxylase（CYP7A1）is a rate-limiting enzyme that breaks down cholesterol into bile acids, which catalyzes cholesterol to produce 7α-hydroxycholesterol, it is an important steroidal drug and intermediate. Taking human CYP7A1 as the research object, firstly, recombinant Pichia pastoris/pPIC3.5K-CYP7A1 was constructed, and then the yield of the target product 7α-hydroxycholesterol increased by site-directed mutation and adaptation to NADPH cytochrome oxidoreductase（CPR）from different sources. Among them, the mutant G485A increased the yield of 7α-hydroxycholesterol by 8.70%. The co-expression of P. pastoris with five different sources of CPR and CYP7A1 increased the yield of 7α-hydroxycholesterol, and the CPR（SgCPR）derived from Siraitia grosvenorii was the most compatible with CYP7A1, which increased the yield of 7α-hydroxycholesterol by 82.26%. Based on the above research, the co-expression of G485A and SgCPR in P. pastoris increased the yield of 7α-hydroxycholesterol by 133.79%, and the yield was 0.25 mg/L. The human cholesterol 7α-hydroxylase was successfully expressed in P. pastoris, and the yield of 7α-hydroxycholesterol increased based on molecular modification of CYP7A1 and adaptation of CPR from different sources.

Key words: steroidal drugs, cholesterol, cholesterol 7α-hydroxylase, 7α-hydroxycholesterol, Pichia pastoris

赵昕, 杜玉瑶, 殷子扬, 毛淑红. 胆固醇7α-羟化酶在毕赤酵母中的异源表达[J]. 生物技术通报, 2023, 39(10): 304-310.

ZHAO Xin, DU Yu-yao, YIN Zi-yang, MAO Shu-hong. Allogeneic Expression of Cholesterol 7α-hydroxylase in Pichia pastoris[J]. Biotechnology Bulletin, 2023, 39(10): 304-310.

图/表 8

图1 胆固醇7α-羟化酶的电子传递机理

Fig. 1 Electron transport mechanism of cholesterol 7α-hydroxylase

表1 CYP7A1基因野生型及突变型引物

Table 1 Wild-type and mutant primers of CYP7A1 gene

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
CYP7A1-F	CGCGGATCCATGATGACCACCTCTCTGATT
CYP7A1-R	CCGGAATTCTTACAGATGTTTAAATTTATATTTAAATTCAAT
H111A-F	AAAGCCTTTGGTGCTAGGTCTATTGATCCAATGGATGGTAATAC
H111A-R	ACCAAAGGCTTTGGCAGAGGT
I114A-F	GGTCATAGGTCTGCTGATCCAATGGATGGTAATACCACTGA
I114A-R	AGACCTATGACCAAAGGCTTTGGC
V281A-F	AAAACCCATCTGGCTGTTCTGTGGGCCTCTCAAGCC
V281A-R	CAGATGGGTTTTGGCTTTTTCC
W284A-F	CTGGTTGTTCTGGCYGCCTCTCAAGCCAATACCATTCC
W284A-R	CAGAACAACCAGATGGGTTTTGG
G485A-F	CAGTCTAGAGCTGCTCTGGGTATTCTGCCACCACTGA
G485A-R	AGCTCTAGACTGATCCAGTGGTGGAC

图2 重组毕赤酵母催化胆固醇TLC结果图混标：胆固醇和7α-羟基胆固醇的混合标品；CYP：CYP7A1（WT）；GS115：毕赤酵母GS115；111：突变体H111A；114：突变体I114A；281：突变体V281A；284：突变体W284A；485：突变体G485A

Fig. 2 TLC analysis of biotransformation result of cholesterol catalyzed by recombinant P. pastoris Mixed labels: Mixed standards of cholesterol and 7α-hydroxycholesterol; CYP: CYP7A1（WT）; GS115: P. pastoris GS115; 111: mutant H111A; 114: mutant I114A; 281: mutant V281A; 284: mutant W284A; 485: mutant G485A

图3 重组毕赤酵母催化生成7α-羟基胆固醇的GC-MS分析图 A：7α-羟基胆固醇质谱图；B：重组毕赤酵母转化产物质谱图

Fig. 3 GC-MS analysis of the 7α-hydroxycholesterol catalyzed by recombinant P. pastoris A: Mass spectra of 7α-hydroxycholesterol. B: Mass spectra of recombinant P. pastoris products

图4 突变毕赤酵母产率比较图突变体H111A、I114A、V281A、W284A和G485A与CYP7A1（WT）重组毕赤酵母的产物比率

Fig. 4 Production abilities of the mutants when compared with the wild type Product ratio of mutant H111A, I114A, V281A, W284A and G485A to CYP7A1（WT）recombinant P. pastoris

图5 CYP7A1与不同来源CPR共表达毕赤酵母对胆固醇转化能力的影响 CYP7A1（WT）与ScCPR、hCPR、AnCPR、RatCPR和SgCPR共表达毕赤酵母，及G485A与SgCPR共表达毕赤酵母产物增长率（与CYP7A1（WT）重组毕赤酵母相比）

Fig. 5 Effects of P. pastoris co-expressed with CYP7A1 and CPR from different sources on cholesterol conversionThe growth rate of products of P. pastoris co-expressed with CYP7A1（WT）and CPR（ScCPR, hCPR, AnCPR, RatCPR and SgCPR）and P. pastoris co-expressed with G485A and SgCPR when compared with that of recombinant P. pastoris containing only CYP7A1（WT）

图6 CYP7A1与胆固醇分子对接结果图黄色部分分子为胆固醇，其他部分为CYP7A1，蓝色标注位点为突变位点

Fig. 6 Docking results of CYP7A1 and cholesterol molecules The yellow molecule is cholesterol, the others are CYP7A1, and the blue-labeled sites are mutation sites

图7 CYP7A1与胆固醇分子对接结果的二维平面图

Fig. 7 2D floor plan of docking results of CYP7A1 and cholesterol molecules

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