赭曲霉11α羟化酶的克隆表达及关键氨基酸位点分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0572

摘要/Abstract

摘要：

11α，17α-双羟基黄体酮是甾体激素类药物的重要中间体，工业上主要利用霉菌对17α-羟基黄体酮的11α羟化反应制备。对赭曲霉的11α羟化酶及其关键氨基酸位点展开研究，为深入解析酶的催化机理提供基础数据。利用底物转化实验探究了10个羟化反应常用霉菌对17α-羟基黄体酮的转化能力，考察了赭曲霉来源的11α羟化酶CYP68J5在不同表达系统中的活性，借助结构预测、分子对接和定点突变等手段对CYP68J5的关键氨基酸位点进行解析。结果表明，赭曲霉的转化能力最强，转化时间60 h的摩尔产率达到最大值，为78.55%；其羟化酶CYP68J5在酿酒酵母中的表达活性最高；位于底物结合口袋附近的D118、F216、M488是CYP68J5的关键氨基酸位点，这些位点在维持酶的结构稳定性上发挥重要作用，是后续分子改造的潜在重要靶点。

关键词: 11α, 17α-双羟基黄体酮, 17α-羟基黄体酮, 赭曲霉, 11α羟化酶, 关键氨基酸位点

Abstract:

11α, 17α-dihydroxy progesterone is an important intermediate of steroid drugs, and is mainly produced by the 11α hydroxylation reaction of 17α-hydroxyprogesterone with molds in industry. In this paper, 11α hydroxylase of Aspergillus ochraceus and its key amino acid sites were investigated, which can provide basic data for further analysis of the catalytic mechanism of 11α hydroxylase. Using the substrate transformation, the conversion of 17α-hydroxyprogesterone by 10 molds with hydroxylation reaction was studied, then the activity of 11α hydroxylase CYP68J5 from A. ochraceus in different expressing systems was evaluated. Finally, the key amino acid sites of CYP68J5 were analyzed by means of structure prediction, molecular docking and site-directed mutation. The results showed that A. ochraceus had the strongest conversion ability, and the maximum molar production rate of 11α, 17α-dihydroxy progesterone reached 78.55% at 60 h. CYP68J5 had the highest activity in Saccharomyces cerevisiae. D118, F216 and M488 near the substrate binding pocket were identified as key amino acid sites of CYP68J5, and they played important roles in maintaining the structural stability of the enzyme and would be the potential targets for further engineering.

Key words: 11α, 17α-dihydroxy progesterone, 17α-hydroxyprogesterone, Aspergillus ochraceus, 11α hydroxylase, key amino acid site

艾露, 陈文慧, 史京辉, 任志远, 沈文琦, 杨嘉凝, 骆健美, 王敏. 赭曲霉11α羟化酶的克隆表达及关键氨基酸位点分析[J]. 生物技术通报, 2023, 39(4): 114-123.

AI Lu, CHEN Wen-hui, SHI Jing-hui, REN Zhi-yuan, SHEN Wen-qi, YANG Jia-ning, LUO Jian-mei, WANG Min. Cloning and Expression of 11α Hydroxylase from Aspergillus ochraceus and Analysis of Key Amino Acid Sites[J]. Biotechnology Bulletin, 2023, 39(4): 114-123.

图/表 11

表1 本实验所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	酶切位点 Restriction site	引物用途 Primer application
11αAO-F	ATGCCCTTCTTCACTGGGCTTCTGGCGAT	无	从赭曲霉cDNA扩增11α羟化酶基因
11αAO-R	CTACACAGTTAAACTCGCCATATCGATCTCCGCC	无	Amplification of 11α hydroxylase gene from cDNA of Aspergillus ochraceus
CYP68J5-F	GCCGCCAGTGTGCTGGAATTCATGCCATTTTTTA- CTGGTTTGTTG	EcoR I	构建酿酒酵母表达CYP68J5重组菌株 Construction of the recombinant S. cerevisiae expressing CYP68J5
CYP68J5-R	TACATGATGCGGCCCTCTAGATTAAACAGTCAA- AGAAGCCATATCAA	Xba I
CYP68J5-F	CGGGATCCCATGAACATCAAGAAGTTCGCCAAG	BamH I	构建毕赤酵母表达CYP68J5重组菌株 Construction of the recombinant P. pastorisexpressing CYP68J5
CYP68J5-R	CGGAATTCTCACTTGTTGACGGTGAGCTGCC	EcoR I
CYP68J5-F	CGGAATTCATGCCGTTCTTCACCGGCC	EcoR I	构建紫红红球菌表达CYP68J5重组菌株
CYP68J5-R	GCTCTAGATCTAGATTAGACGGTGAGCGAGGCC	Xba I	Construction of the recombinant R. rhodochrous expressing CYP68J5
CYP68J5_W89A-F	GCGGGTTCTTTGATTGTTTTGCCAC	无	构建CYP68J5突变体
CYP68J5_W89A-R	TTCAGACATCAATCTATATGGTTCATGAC	无	Construction of mutants of CYP68J5
CYP68J5_F111A-F	GCTGAAACTCCAACTACTGATGATTCTC	无
CYP68J5_F111 A-R	ATCCATTCTTGGATCATTTCTCAATTC	无
CYP68J5_T115A-F	GCTACTGATGATTCTCATGGTTATATTCC	无
CYP68J5_T115A-R	TGGAGTTTCAAAATCCATTCTTGGATC	无
CYP68J5_D118A-F	GCTTCTCATGGTTATATTCCTGGTTTTG	无
CYP68J5_D118A-R	ATCAGTAGTTGGAGTTTCAAAATCCAT	无
CYP68J5_S119A-F	GCTCATGGTTATATTCCTGGTTTTG	无
CYP68J5_S119A-R	ATCATCAGTAGTTGGAGTTTCAAAATC	无
CYP68J5_L129A-F	GCAAATGCTGATCCAAATTTGACTAAAG	无
CYP68J5_L129A-R	AGCATCAAAACCAGGAATATAACCATG	无
CYP68J5_F216A-F	GCTGGTGTTGGTGATAAATTGAGAAT	无
CYP68J5_F216A-R	AGCCAAAGCAGCATATTGAGAAGAAGTT	无
CYP68J5_R223A-F	GCAATTTATCCAAGAATGATTAGACC	无
CYP68J5_R223A-R	CAATTTATCACCAACACCAAAAGCC	无
CYP68J5_I303A-F	GCTGTTGCTATTCATACTACTTCTG	无
CYP68J5_I303A-R	AGACAAAGTAACTTGTTTCAAAACAG	无
CYP68J5_V304A-F	GCTGCTATTCATACTACTTCTGATTTGT	无
CYP68J5_V304A-R	AATAGACAAAGTAACTTGTTTCAAAAC	无
CYP68J5_T308A-F	GCTACTTCTGATTTGTTATTGCAAGCT	无
CYP68J5_T308A-R	ATGAATAGCAACAATAGACAAAGTAAC	无
CYP68J5_L368A-F	GCGTTGGGATCTTTCAGAAGACAAG	无
CYP68J5_L368A-R	AGTTGGTCTCAATCTTTGAGATTCTTTC	无
CYP68J5_R373A-F	GCAAGACAAGCTACTAATGATATTAAATT	无
CYP68J5_R373A-R	GAAAGATCCCAACAAAGTTGGTCTC	无
CYP68J5_M488A-F	GCGACTTATTTGGCTGATCCAAACAC	无
CYP68J5_M488A-R	ACCAATATTCAATGGTTGTGGTTTAAAAC	无
CYP68J5_T489A-F	GCTTATTTGGCTGATCCAAACACTAG	无
CYP68J5_T489A-R	CATACCAATATTCAATGGTTGTGGTTT	无

表1 本实验所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	酶切位点 Restriction site	引物用途 Primer application
11αAO-F	ATGCCCTTCTTCACTGGGCTTCTGGCGAT	无	从赭曲霉cDNA扩增11α羟化酶基因
11αAO-R	CTACACAGTTAAACTCGCCATATCGATCTCCGCC	无	Amplification of 11α hydroxylase gene from cDNA of Aspergillus ochraceus
CYP68J5-F	GCCGCCAGTGTGCTGGAATTCATGCCATTTTTTA- CTGGTTTGTTG	EcoR I	构建酿酒酵母表达CYP68J5重组菌株 Construction of the recombinant S. cerevisiae expressing CYP68J5
CYP68J5-R	TACATGATGCGGCCCTCTAGATTAAACAGTCAA- AGAAGCCATATCAA	Xba I
CYP68J5-F	CGGGATCCCATGAACATCAAGAAGTTCGCCAAG	BamH I	构建毕赤酵母表达CYP68J5重组菌株 Construction of the recombinant P. pastorisexpressing CYP68J5
CYP68J5-R	CGGAATTCTCACTTGTTGACGGTGAGCTGCC	EcoR I
CYP68J5-F	CGGAATTCATGCCGTTCTTCACCGGCC	EcoR I	构建紫红红球菌表达CYP68J5重组菌株
CYP68J5-R	GCTCTAGATCTAGATTAGACGGTGAGCGAGGCC	Xba I	Construction of the recombinant R. rhodochrous expressing CYP68J5
CYP68J5_W89A-F	GCGGGTTCTTTGATTGTTTTGCCAC	无	构建CYP68J5突变体
CYP68J5_W89A-R	TTCAGACATCAATCTATATGGTTCATGAC	无	Construction of mutants of CYP68J5
CYP68J5_F111A-F	GCTGAAACTCCAACTACTGATGATTCTC	无
CYP68J5_F111 A-R	ATCCATTCTTGGATCATTTCTCAATTC	无
CYP68J5_T115A-F	GCTACTGATGATTCTCATGGTTATATTCC	无
CYP68J5_T115A-R	TGGAGTTTCAAAATCCATTCTTGGATC	无
CYP68J5_D118A-F	GCTTCTCATGGTTATATTCCTGGTTTTG	无
CYP68J5_D118A-R	ATCAGTAGTTGGAGTTTCAAAATCCAT	无
CYP68J5_S119A-F	GCTCATGGTTATATTCCTGGTTTTG	无
CYP68J5_S119A-R	ATCATCAGTAGTTGGAGTTTCAAAATC	无
CYP68J5_L129A-F	GCAAATGCTGATCCAAATTTGACTAAAG	无
CYP68J5_L129A-R	AGCATCAAAACCAGGAATATAACCATG	无
CYP68J5_F216A-F	GCTGGTGTTGGTGATAAATTGAGAAT	无
CYP68J5_F216A-R	AGCCAAAGCAGCATATTGAGAAGAAGTT	无
CYP68J5_R223A-F	GCAATTTATCCAAGAATGATTAGACC	无
CYP68J5_R223A-R	CAATTTATCACCAACACCAAAAGCC	无
CYP68J5_I303A-F	GCTGTTGCTATTCATACTACTTCTG	无
CYP68J5_I303A-R	AGACAAAGTAACTTGTTTCAAAACAG	无
CYP68J5_V304A-F	GCTGCTATTCATACTACTTCTGATTTGT	无
CYP68J5_V304A-R	AATAGACAAAGTAACTTGTTTCAAAAC	无
CYP68J5_T308A-F	GCTACTTCTGATTTGTTATTGCAAGCT	无
CYP68J5_T308A-R	ATGAATAGCAACAATAGACAAAGTAAC	无
CYP68J5_L368A-F	GCGTTGGGATCTTTCAGAAGACAAG	无
CYP68J5_L368A-R	AGTTGGTCTCAATCTTTGAGATTCTTTC	无
CYP68J5_R373A-F	GCAAGACAAGCTACTAATGATATTAAATT	无
CYP68J5_R373A-R	GAAAGATCCCAACAAAGTTGGTCTC	无
CYP68J5_M488A-F	GCGACTTATTTGGCTGATCCAAACAC	无
CYP68J5_M488A-R	ACCAATATTCAATGGTTGTGGTTTAAAAC	无
CYP68J5_T489A-F	GCTTATTTGGCTGATCCAAACACTAG	无
CYP68J5_T489A-R	CATACCAATATTCAATGGTTGTGGTTT	无

图1 标准品（17α-羟基黄体酮和11α，17α-双羟基黄体酮）（a）及转化样品（b）的HPLC图谱

Fig.1 HPLC profiles of standards（17α-hydroxyprogest-erone and 11α，17α-dihydroxy progesterone）（a）and biotransformation sample（b）

图2 10个霉菌转化17α-羟基黄体酮48 h的TLC结果 1：17α-羟基黄体酮标准品；2：11α，17α-双羟基黄体酮标准品；3：赭曲霉；4：雅致小克银汉霉；5：蓝色犁头霉；6：球孢白僵菌；7：金龟子绿僵菌；8：黑曲霉；9：黄绿青霉；10：黄曲霉；11：桔青霉；12：雷斯青霉

Fig. 2 TLC results of 17α-hydroxyprogesterone at 48 h by 10 molds 1：Standard of 17α-hydroxyprogesterone. 2：Standard of 11α，17α-dihydroxy progesterone. 3：Aspergillus ochraceus. 4：Cunningpamycetes elegans. 5：Absidia coerulea. 6：Beauveria bassiana. 7：Metarhizium anisopliae. 8：Aspergillus niger. 9：Penicillium citreoviridin. 10：Aspergillus flavus. 11：Penicillium citrinum. 12：Penicillium raistrickii

图3 赭曲霉和雅致小克银汉霉转化17α-羟基黄体酮生成11α，17α-双羟基黄体酮的摩尔产率（a）和生产强度（b）

Fig. 3 Molar production rate（a）and production intensity（b）of 11α，17α-dihydroxy progesterone from 17α-hydroxyprogesterone by Aspergillus ochraceus and Cunningpamycetes elegans，respectively

图4 表达CYP68J5的毕赤酵母重组菌株（a）、酿酒酵母重组菌株（b）和紫红红球菌重组菌株（c）验证

Fig. 4 Verification of the recombinant P. pastoris（a），S. cerevisiae（b）and R. rhodochrous（c）expressing CYP68J5 M：DL 2000 DNA marker；1：PCR product of pYES2-11αAo；2：PCR product of pNV18-11αAo

图5 表达CYP68J5的酿酒酵母、毕赤酵母和紫红红球菌重组菌株转化17α-羟基黄体酮48 h的TLC结果 1：17α-羟基黄体酮标准品；2：11α，17α-双羟基黄体酮标准品；3：酿酒酵母；4：毕赤酵母；5：紫红红球菌

Fig. 5 TLC biotransformation results of 17α-hydroxypro-gesterone at 48 h by the recombinant S. cerevisiae，P. pastoris and R. rhodochrous expressing CYP68J5，respectively 1：Standard of 17α-hydroxyprogesterone. 2. Standard of 11α，17α-dihydroxy progesterone. 3：Saccharomyces cerevisiae. 4：Pichia pastoris. 5：Rhodococcus rhodochrous

图6 表达CYP68J5酿酒酵母和毕赤酵母重组菌株转化17α-羟基黄体酮生成11α, 17α-双羟基黄体酮的摩尔产率（a）和生产强度（b）

Fig. 6 Molar production rate（a）and production inten-sity（b）of 11α, 17α-dihydroxy progesterone from 17α-hydroxyprogesterone by the recombinant S. cer-evisiae and P. pastoris expressing CYP68J5, respec-tively

图7 表达CYP68J5的系列酿酒酵母重组菌株的转化性能

Fig. 7 Biotransformation performance of recombinant S. cerevisiae strains expressing CYP68J5

图8 预测的CYP68J5总体结构及其与17α-羟基黄体酮的分子对接 α螺旋、β折叠和无规则卷曲分别用亮蓝色、亮紫色和粉红色表示，活性位点和底物分别用亮黄色和深红色表示

Fig. 8 Predicted overall structure of CYP68J5 and the molecular docking with 17α-hydroxyprogesterone α helix，β folding and random curl are colored in bright blue，bright purple and pink，respectively. The active site residues and the substrate are colored in bright yellow and dark red，respectively

图9 CYP68J5野生型及其突变体转化17α-羟基黄体酮生成11α, 17α-双羟基黄体酮的相对摩尔产率数据代表3个独立实验的平均值。以CYP68J5野生型的摩尔产率为100%计算相对摩尔产率

Fig. 9 Relative molar production rate of 11α, 17α-dihydr-oxy progesterone from 17α-hydroxyprogesterone by the mutants compared to that of the wild-type CYP68J5 The data represent the mean value of three independent experiments. The relative molar production rate is calculated by using the molar production rate of wild-type CYP68J5 as 100%

图10 CYP68J5的关键氨基酸位点D118（a）、F216（b）和M488（c）及突变为丙氨酸后A118（d）、A216（e）和A488（f）局部模型

Fig. 10 Local model of the key amino acid site residues D118（a），F216（b）and M488（c）of CYP68J5 and the alanine mutation site residues A118（d），A216（e）and A488（f）

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