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Article(id=1200500176222810534, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-1325, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1700755200000, receivedDateStr=2023-11-24, revisedDate=1705334400000, revisedDateStr=2024-01-16, acceptedDate=null, acceptedDateStr=null, onlineDate=1764151146225, onlineDateStr=2025-11-26, pubDate=1718121600000, pubDateStr=2024-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764151146225, onlineIssueDateStr=2025-11-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764151146225, creator=13701087609, updateTime=1764151146225, updator=13701087609, issue=Issue{id=1200500165426672625, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='6', pageStart='1509', pageEnd='1896', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764151143651, creator=13701087609, updateTime=1764225143180, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810542001680840, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810542001680841, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1883, endPage=1893, ext={EN=ArticleExt(id=1200500176835178970, articleId=1200500176222810534, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Study on the catalytic mechanism of triterpene C-29 carboxylases from Tripterygium wilfordii based on directed evolution, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng, which have anti-tumour, anti-inflammatory and immunosuppressive activities, and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations. By analysing the biosynthetic pathway of active ingredients, optimizing genetic elements and utilizing "cell factory" to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a "low-cost and high-efficient" manner. CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T. wilfordii, and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid from β-amyrin by carboxylation at C-29 position. In this study, four multifunctional TwCYP712K1/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae. The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking, and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure. A total of 63 mutant elements were constructed, and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed. In particular, the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2F127I and TwCYP712K2A227T mutants would result in a reversal of the product ratio. In conclusion, four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins, and a series of activity-enhancing or altering mutants were obtained, which provide abundant catalytic elements for the biosynthesis of active triterpenoids from T. wilfordii, and the mechanism of carboxylation in the C-29 position was initially elucidated.

, correspAuthors=Xia-nan ZHANG, Wei GAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Pan-ting LIU, Yi-feng ZHANG, Yuan LIU, Jie GAO, Lin MA, Xiao-yi WU, Ya-ting HU, Ping SU, Shi-jun YUAN, Xia-nan ZHANG, Wei GAO), CN=ArticleExt(id=1200500180010267330, articleId=1200500176222810534, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于定向进化的雷公藤三萜C-29位羧化酶催化机制研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

雷公藤红素和雷公藤内酯甲是中药雷公藤的主要三萜活性成分, 具有抗肿瘤、抗炎及免疫抑制等药理活性, 是雷公藤相关中药制剂发挥临床疗效的物质基础。通过解析活性成分生源途径, 优化基因元件并利用“细胞工厂”异源获取将是未来“低成本、高效率”获得雷公藤中三萜活性成分的有效途径。CYP712Ks是参与雷公藤木栓烷型和齐墩果烷型三萜骨架修饰的第一个细胞色素P450酶, 发挥C-29位羧基化功能, 可催化木栓酮生成美登木酸, 催化β-香树素生成3-表卡通酸。本研究以四条多功能TwCYP712K1/2/3/5为研究对象, 利用酿酒酵母体内功能表征, 明确其催化功能及底物选择偏好性; 通过同源建模和分子对接, 明确蛋白与底物结合空间结构, 进而对蛋白活性口袋内差异氨基酸进行互相突变, 阐明决定催化功能和底物结构选择的关键氨基酸。本研究共构建了63个突变体元件, 解析了影响TwCYP712Ks羧基化功能的氨基酸位点, 特别是揭示了影响TwCYP712K2对齐墩果烷型和木栓烷型三萜底物选择性的关键氨基酸, TwCYP712K2F127I与TwCYP712K2A227T突变体将导致产物比例发生逆转。本研究以同源蛋白比对及相互突变的方式对TwCYP712Ks四条蛋白进行半理性设计, 阐明多个决定蛋白催化功能的氨基酸位点, 获得系列活性提升或改变的突变体, 为雷公藤三萜活性成分的生物合成提供丰富的催化元件, 并初步解析了C-29位羧基化机制。

, correspAuthors=张夏楠, 高伟, authorNote=null, correspAuthorsNote=
*张夏楠, E-mail: ;
高伟, Tel: 86-10-83911633, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=MkclIlh9NX2xG6FBxWfilw==, magXml=8gKuc4XgFmafpWvZQ97hew==, pdfUrl=null, pdf=AewdZPeqa0TUkkgxr53CKQ==, pdfFileSize=7913986, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=PA4L2j8PZl3IP2fsxkNpEg==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=NyGBoR78IZ5W4RTFvqWEEA==, mapNumber=null, authorCompany=null, fund=null, authors=

#共同第一作者.

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National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1201118426346512727, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, authorId=1201118426078077259, language=CN, stringName=张逸风, firstName=逸风, middleName=null, lastName=张, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=2, #, address=2.道地药材品质保障与资源持续利用全国重点实验室, 中国中医科学院中药资源中心, 北京 100700, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1201118425432154408, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, xref=null, ext=[AuthorCompanyExt(id=1201118425448931626, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, companyId=1201118425432154408, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2. 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Controlling chemo- and regioselectivity of a plant P450 in yeast cell toward rare licorice triterpenoid biosynthesis [J]. ACS Catal, 2020, 10: 4253-4260., articleTitle=null, refAbstract=null), Reference(id=1201118443274723546, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[16], rfOrder=15, authorNames=null, journalName=null, refType=null, unstructuredReference=Xu H, Dickschat JS. Turning a sesquiterpene synthase into a di- and sesterterpene synthase [J]. 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Angew Chem Int Ed Engl, 2022, 61: e202113587., articleTitle=null, refAbstract=null)], funds=[Fund(id=1201118439000727598, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, awardId=81973418, language=CN, fundingSource=国家自然科学基金面上项目(81973418), fundOrder=null, country=null), Fund(id=1201118439151722554, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, awardId=81974515, language=CN, fundingSource=国家自然科学基金面上项目(81974515), fundOrder=null, country=null), Fund(id=1201118439290134593, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, awardId=2020YFA0908000, language=CN, fundingSource=国家重点研发计划项目(2020YFA0908000), fundOrder=null, country=null), Fund(id=1201118439428546630, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, awardId=ZYYCXTD-D-202005, language=CN, fundingSource=国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1201118425226633505, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, xref=null, ext=[AuthorCompanyExt(id=1201118425239216419, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, companyId=1201118425226633505, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. 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Colour key: CYP712K that mainly catalysing the oxidation of the C-29 position of the friedelane-type triterpene are labelled red and those mainly catalysing the oxidation of the C-29 position of the oleanane-type triterpene are labelled blue , figureFileSmall=gSu/sDtOzvdo2DMsJBZiGw==, figureFileBig=prNx1UCbHSUD9WTWusGnUA==, tableContent=null), ArticleFig(id=1201118436031161277, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=dqqeJiAhAwbEj/30fANPjg==, figureFileBig=1dqzjCPffMB1icb5P4tY0A==, tableContent=null), ArticleFig(id=1201118436144407497, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Figure 2, caption= 　Construction of chassis strain and comparison of TwCYP712Ks catalytic activity. A: Construction strategy of the BY-SH yeast strain for friedelin and <i>β</i>-amyrin productions by CRISPR-Cas9 method; B: GC-MS analysis of the fermentation products of BY-SH; C: Mass spectrum of the products of BY-SH; D: Relative yields of 3-<i>epi</i>-katonic acid and polpunonic acid in yeast expressing <i>TwCYP712Ks</i>. Relative content represents the amount of all metabolites relative to the amount of 3-<i>epi</i>-katonic acid produced by TwCYP712K1. <i>n</i> <i>=</i> 3, <span class="mag-xml-inline-formula"><tex-math id="M1">$ \stackrel{-}{x} $</tex-math></span> ± <i>s</i>. <sup><i>***</i></sup><i>P</i> < 0.001, <sup><i>****</i></sup><i>P</i> < 0.000 1 <i>vs</i> TwCYP712K1; E: LC-MS analysis of BY-SH products expressing <i>TwCYP712Ks</i>; F, G: Mass spectrum of 3-<i>epi</i>-katonic acid (F) and polpunonic acid (G) produced by TwCYP712Ks , figureFileSmall=dqqeJiAhAwbEj/30fANPjg==, figureFileBig=1dqzjCPffMB1icb5P4tY0A==, tableContent=null), ArticleFig(id=1201118436282819536, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=M72oRAVBNiJ980e0/H534A==, figureFileBig=nFloIZbPHMHLYfnpbv3OPw==, tableContent=null), ArticleFig(id=1201118436429620184, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Figure 3, caption= 　Bioinformatics analysis of TwCYP712Ks. A: Multiple sequence analysis of TwCYP712Ks. The yellow triangle marks the selected amino acid sites for mutation; B: Motif analysis of TwCYP712Ks , figureFileSmall=M72oRAVBNiJ980e0/H534A==, figureFileBig=nFloIZbPHMHLYfnpbv3OPw==, tableContent=null), ArticleFig(id=1201118436593198051, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=38k4EHJYAxO4OcJhn64rlw==, figureFileBig=DBRPupm0lF7kIVyla/9ARg==, tableContent=null), ArticleFig(id=1201118436731610092, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Figure 4, caption= 　Homology modeling and molecular docking analysis of TwCYP712Ks protein and amino acid residues in the active pocket selected for mutagenesis. A-D: The docking results of TwCYP712Ks with friedelin; E-H: The docking results of TwCYP712Ks with <i>β</i>-amyrin. The displayed amino acid residues are 5 Å away from the substrate , figureFileSmall=38k4EHJYAxO4OcJhn64rlw==, figureFileBig=DBRPupm0lF7kIVyla/9ARg==, tableContent=null), ArticleFig(id=1201118436844856308, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=J8FsQV4fZF12yjF+KVrUZQ==, figureFileBig=ULP6EaEESeWxHMRXKklSpQ==, tableContent=null), ArticleFig(id=1201118438048621561, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Figure 5, caption= 　Mutagenesis assay of TwCYP712Ks and molecular docking. A: Relative yield of polpunonic acid in yeast harboring TwCYP712K1 or its mutants; B: Relative yields of the products in yeast harboring TwCYP712K2 or its mutants and the active site of TwCYP712K2. The HEM is shown in green, while the substrates friedelin and <i>β</i>-amyrin are shown in yellow and pink, respectively. The residues that can enhance or reduce protein activity are shown in blue or rose, respectively; C: Relative yields of the products in yeast harboring TwCYP712K3 or its mutants; D: Relative yields of the products in yeast harboring TwCYP712K5 or its mutants. All data were analyzed based on the wild-type yield of each gene as 100% , figureFileSmall=J8FsQV4fZF12yjF+KVrUZQ==, figureFileBig=ULP6EaEESeWxHMRXKklSpQ==, tableContent=null), ArticleFig(id=1201118438203809796, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namePrimer sequence (5′-3′)
X2-up-FCGTCTATGAGGAGACTGTTAGTTGGAT
X2-up-hr-RCTTTGAAATGGCAGTATTGATAATGATAAACTCGAGACCACTTCGAGAGCAAGTTGCCTG
TDH3p-FTCGAGTTTATCATTATCAATACTGCCATTTCAAAG
TDH3p-hr-RCTCAGCAACTTTGAGCTTCCACATTTTGTTTGTTTATGTGTGTTTATTCG
TwOSC1T502Eopt-FATGTGGAAGCTCAAAGTTGCTGAG
TwOSC1T502Eopt-hr-RCACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTCAATAGCCTTTGGAT
ADH1t-FGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG
ADH1t-hr-RGGCCCGAAATTGTTCCTACGAGGCATATCTACAATTGGGTGAAATGG
tHXT7p-FCTCGTAGGAACAATTTCGGGCC
tHXT7p-hr-RCCGCTATCTTCAGCCTCCACATTTTTTGATTAAAATTAAAAAAACTTTTTGTTT
GgbAS1-FATGTGGAGGCTGAAGATAGCGG
GgbAS1-hr-RTCTAAACAGATTCAATACTCATTAAAAAACTATATCAATTAATTTGAATTAACTTAAGTTAAACAAACTG
FBA1t-FGTTAATTCAAATTAATTGATATAGTTTTTTAATGAGTATTGAATCTGTTTAGA
FBA1t-hr-RCTATTGTGCAGATGTTATAATATCTGTGCGTAGTAAGCTACTATGAAAGACTTTAC
PGK1p-FACGCACAGATATTATAACATCTGCACAATAG
PGK1p-hr-RGAACTAGGTCAGAGCTGCTAGACATTTTGTTATATTTGTTGTAAAAAGTAGATAATTA
TwCPR3opt-FATGTCTAGCAGCTCTGACCTAGTTC
TwCPR3opt-hr-RGCATGAATTATTAATAATAAAAACTAAATCATTAAAGTAACTTAAGGAGTTAAATTTACCAAACATCCCTCA
TDH2t-FATTTAACTCCTTAAGTTACTTTAATGATTTAGTTTTTATTATTAATAATTCATGC
TDH2t-hr-RTCTGTGAGGCCGATTATGCAGGGCGAAAAGCCAATTAGTGTG
X2-down-FCCTGCATAATCGGCCTCACAGA
X2-down-RCTCGCCAAGGCATTACCATCCC
PCR1-F/RTCTCAGATGAGGACAATGTAGTGGTAG/ TCCTCGGTGAACTTGCGACGAGCCTC
PCR2-F/RGGTGGTTACCCCCAACAGGAAATCACTGGAG/ CCACAGGGCTTGACCTGGAAGCGGT
PCR3-F/RGAGACCCTGCTCCTCTTCATCGAGCTGC/ CAGACAATGGCTTGGGCACGAATACGGG
PCR4-F/RGTTTGCGGCGACGCGAAAGGTATGGC/ GTAACAGCAAAAGACGCCACCA
p416-CYP712K1-FTTTCTAGAACTAGTGGATCCATGGCCACCATCACTGACATC
p416-CYP712K1-RACATGACTCGAGGTCGACGGTTAACCGGCAAATGGATTGAAAC
p416-CYP712K2-FTTTCTAGAACTAGTGGATCCATGGCCACTACCATCATTG
p416-CYP712K2-RACATGACTCGAGGTCGACGGTTAGGAAGAAAATGGATCAAATC
p416-CYP712K3-FTTTCTAGAACTAGTGGATCCATGGCCACCACTACCATC
p416-CYP712K3-RACATGACTCGAGGTCGACGGTTAGCAAGAAAAGGGATGGAATC
p416-CYP712K5-FTTTCTAGAACTAGTGGATCCATGACAACAATCACTGATGT
p416-CYP712K5-RACATGACTCGAGGTCGACGGTTAAGAAGAAAATGGATTGAACC
), ArticleFig(id=1201118438375776270, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Table 1, caption=

　The primers used in this study. F: Forward; R: Reverse

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namePrimer sequence (5′-3′)
X2-up-FCGTCTATGAGGAGACTGTTAGTTGGAT
X2-up-hr-RCTTTGAAATGGCAGTATTGATAATGATAAACTCGAGACCACTTCGAGAGCAAGTTGCCTG
TDH3p-FTCGAGTTTATCATTATCAATACTGCCATTTCAAAG
TDH3p-hr-RCTCAGCAACTTTGAGCTTCCACATTTTGTTTGTTTATGTGTGTTTATTCG
TwOSC1T502Eopt-FATGTGGAAGCTCAAAGTTGCTGAG
TwOSC1T502Eopt-hr-RCACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTCAATAGCCTTTGGAT
ADH1t-FGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG
ADH1t-hr-RGGCCCGAAATTGTTCCTACGAGGCATATCTACAATTGGGTGAAATGG
tHXT7p-FCTCGTAGGAACAATTTCGGGCC
tHXT7p-hr-RCCGCTATCTTCAGCCTCCACATTTTTTGATTAAAATTAAAAAAACTTTTTGTTT
GgbAS1-FATGTGGAGGCTGAAGATAGCGG
GgbAS1-hr-RTCTAAACAGATTCAATACTCATTAAAAAACTATATCAATTAATTTGAATTAACTTAAGTTAAACAAACTG
FBA1t-FGTTAATTCAAATTAATTGATATAGTTTTTTAATGAGTATTGAATCTGTTTAGA
FBA1t-hr-RCTATTGTGCAGATGTTATAATATCTGTGCGTAGTAAGCTACTATGAAAGACTTTAC
PGK1p-FACGCACAGATATTATAACATCTGCACAATAG
PGK1p-hr-RGAACTAGGTCAGAGCTGCTAGACATTTTGTTATATTTGTTGTAAAAAGTAGATAATTA
TwCPR3opt-FATGTCTAGCAGCTCTGACCTAGTTC
TwCPR3opt-hr-RGCATGAATTATTAATAATAAAAACTAAATCATTAAAGTAACTTAAGGAGTTAAATTTACCAAACATCCCTCA
TDH2t-FATTTAACTCCTTAAGTTACTTTAATGATTTAGTTTTTATTATTAATAATTCATGC
TDH2t-hr-RTCTGTGAGGCCGATTATGCAGGGCGAAAAGCCAATTAGTGTG
X2-down-FCCTGCATAATCGGCCTCACAGA
X2-down-RCTCGCCAAGGCATTACCATCCC
PCR1-F/RTCTCAGATGAGGACAATGTAGTGGTAG/ TCCTCGGTGAACTTGCGACGAGCCTC
PCR2-F/RGGTGGTTACCCCCAACAGGAAATCACTGGAG/ CCACAGGGCTTGACCTGGAAGCGGT
PCR3-F/RGAGACCCTGCTCCTCTTCATCGAGCTGC/ CAGACAATGGCTTGGGCACGAATACGGG
PCR4-F/RGTTTGCGGCGACGCGAAAGGTATGGC/ GTAACAGCAAAAGACGCCACCA
p416-CYP712K1-FTTTCTAGAACTAGTGGATCCATGGCCACCATCACTGACATC
p416-CYP712K1-RACATGACTCGAGGTCGACGGTTAACCGGCAAATGGATTGAAAC
p416-CYP712K2-FTTTCTAGAACTAGTGGATCCATGGCCACTACCATCATTG
p416-CYP712K2-RACATGACTCGAGGTCGACGGTTAGGAAGAAAATGGATCAAATC
p416-CYP712K3-FTTTCTAGAACTAGTGGATCCATGGCCACCACTACCATC
p416-CYP712K3-RACATGACTCGAGGTCGACGGTTAGCAAGAAAAGGGATGGAATC
p416-CYP712K5-FTTTCTAGAACTAGTGGATCCATGACAACAATCACTGATGT
p416-CYP712K5-RACATGACTCGAGGTCGACGGTTAAGAAGAAAATGGATTGAACC
), ArticleFig(id=1201118438514188310, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Protein pairwise comparisonAmino acid sequence identity
CYP712K1-CYP712K273.35%
CYP712K1-CYP712K372.82%
CYP712K1-CYP712K570.96%
CYP712K2-CYP712K390.27%
CYP712K2-CYP712K576.36%
CYP712K3-CYP712K574.47%
), ArticleFig(id=1201118438644211739, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176222810534, language=CN, label=Table 2, caption=

　TwCYP712Ks amino acid sequence identity

, figureFileSmall=null, figureFileBig=null, tableContent=
Protein pairwise comparisonAmino acid sequence identity
CYP712K1-CYP712K273.35%
CYP712K1-CYP712K372.82%
CYP712K1-CYP712K570.96%
CYP712K2-CYP712K390.27%
CYP712K2-CYP712K576.36%
CYP712K3-CYP712K574.47%
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基于定向进化的雷公藤三萜C-29位羧化酶催化机制研究
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刘攀婷 1, # , 张逸风 2, # , 刘远 1 , 高杰 1 , 马林 1 , 吴晓毅 1 , 胡雅婷 1 , 苏平 2 , 袁仕君 2 , 张夏楠 1, * , 高伟 1, *
药学学报 | 研究论文 2024,59(6): 1883-1893
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药学学报 | 研究论文 2024, 59(6): 1883-1893
基于定向进化的雷公藤三萜C-29位羧化酶催化机制研究
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刘攀婷1, #, 张逸风2, #, 刘远1, 高杰1, 马林1, 吴晓毅1, 胡雅婷1, 苏平2, 袁仕君2, 张夏楠1, * , 高伟1, *
作者信息
  • 1.首都医科大学中医药学院, 北京 100069
  • 2.道地药材品质保障与资源持续利用全国重点实验室, 中国中医科学院中药资源中心, 北京 100700

通讯作者:

*张夏楠, E-mail: ;
高伟, Tel: 86-10-83911633, E-mail:
Study on the catalytic mechanism of triterpene C-29 carboxylases from Tripterygium wilfordii based on directed evolution
Pan-ting LIU1, Yi-feng ZHANG2, Yuan LIU1, Jie GAO1, Lin MA1, Xiao-yi WU1, Ya-ting HU1, Ping SU2, Shi-jun YUAN2, Xia-nan ZHANG1, * , Wei GAO1, *
Affiliations
  • 1. School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China
  • 2. National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2023-1325
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摘要
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雷公藤红素和雷公藤内酯甲是中药雷公藤的主要三萜活性成分, 具有抗肿瘤、抗炎及免疫抑制等药理活性, 是雷公藤相关中药制剂发挥临床疗效的物质基础。通过解析活性成分生源途径, 优化基因元件并利用“细胞工厂”异源获取将是未来“低成本、高效率”获得雷公藤中三萜活性成分的有效途径。CYP712Ks是参与雷公藤木栓烷型和齐墩果烷型三萜骨架修饰的第一个细胞色素P450酶, 发挥C-29位羧基化功能, 可催化木栓酮生成美登木酸, 催化β-香树素生成3-表卡通酸。本研究以四条多功能TwCYP712K1/2/3/5为研究对象, 利用酿酒酵母体内功能表征, 明确其催化功能及底物选择偏好性; 通过同源建模和分子对接, 明确蛋白与底物结合空间结构, 进而对蛋白活性口袋内差异氨基酸进行互相突变, 阐明决定催化功能和底物结构选择的关键氨基酸。本研究共构建了63个突变体元件, 解析了影响TwCYP712Ks羧基化功能的氨基酸位点, 特别是揭示了影响TwCYP712K2对齐墩果烷型和木栓烷型三萜底物选择性的关键氨基酸, TwCYP712K2F127I与TwCYP712K2A227T突变体将导致产物比例发生逆转。本研究以同源蛋白比对及相互突变的方式对TwCYP712Ks四条蛋白进行半理性设计, 阐明多个决定蛋白催化功能的氨基酸位点, 获得系列活性提升或改变的突变体, 为雷公藤三萜活性成分的生物合成提供丰富的催化元件, 并初步解析了C-29位羧基化机制。

雷公藤  /  CYP712K  /  定点突变  /  美登木酸  /  3-表卡通酸

Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng, which have anti-tumour, anti-inflammatory and immunosuppressive activities, and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations. By analysing the biosynthetic pathway of active ingredients, optimizing genetic elements and utilizing "cell factory" to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a "low-cost and high-efficient" manner. CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T. wilfordii, and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid from β-amyrin by carboxylation at C-29 position. In this study, four multifunctional TwCYP712K1/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae. The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking, and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure. A total of 63 mutant elements were constructed, and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed. In particular, the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2F127I and TwCYP712K2A227T mutants would result in a reversal of the product ratio. In conclusion, four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins, and a series of activity-enhancing or altering mutants were obtained, which provide abundant catalytic elements for the biosynthesis of active triterpenoids from T. wilfordii, and the mechanism of carboxylation in the C-29 position was initially elucidated.

Tripterygium wilfordii  /  CYP712K  /  site-directed mutagenesis  /  polpunonic acid  /  3-epi-katonic acid
刘攀婷, 张逸风, 刘远, 高杰, 马林, 吴晓毅, 胡雅婷, 苏平, 袁仕君, 张夏楠, 高伟. 基于定向进化的雷公藤三萜C-29位羧化酶催化机制研究. 药学学报, 2024 , 59 (6) : 1883 -1893 . DOI: 10.16438/j.0513-4870.2023-1325
Pan-ting LIU, Yi-feng ZHANG, Yuan LIU, Jie GAO, Lin MA, Xiao-yi WU, Ya-ting HU, Ping SU, Shi-jun YUAN, Xia-nan ZHANG, Wei GAO. Study on the catalytic mechanism of triterpene C-29 carboxylases from Tripterygium wilfordii based on directed evolution[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1883 -1893 . DOI: 10.16438/j.0513-4870.2023-1325
正文
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雷公藤, 又名断肠草、黄藤等, 是我国传统中药, 来源于卫矛科多年生藤本植物雷公藤 (Tripterygium wilfordii Hook. f.) 的干燥根或根的木质部。味苦、辛, 性寒, 归肝、肾经, 具有祛风除湿、杀虫解毒、消肿止痛、活血通络的功效[1]。雷公藤化学成分繁多且复杂, 自1936年[2]首次从雷公藤根部提取到雷公藤红素 (celastrol), 至今已分离并鉴定出450多种化合物, 主要包括二萜类、三萜类、倍半萜类和生物碱类等。雷公藤中含有丰富的三萜化合物, 如雷公藤红素、雷公藤内酯甲 (wilforlide A) 等, 其中雷公藤红素作为一种天然木栓烷型三萜化合物, 2007年被《Cell》杂志列为最有可能发展为药物的5种天然产物之一[3], 其具有显著的抗炎[4]、抗肿瘤[5]、抗肥胖[6]和抗中枢神经系统疾病[7]等药理活性, 而雷公藤内酯甲同样具有良好的抗炎及免疫抑制等作用[8,9]。目前雷公藤药品或制剂中的三萜类成分主要来源于植物提取, 然而雷公藤植物生长周期长, 化合物积累量低导致提取困难, 传统的栽培方法难以满足日益增长的市场需求; 化学全合成或半合成的步骤复杂、成本高且对环境不友好。近年来, 药用天然产物生物合成和合成生物学领域的研究不断取得新突破, 通过在微生物宿主中重构生物合成途径来实现雷公藤红素和雷公藤内酯甲的异源获取已成为可能[10]。这种经人工设计构建的可按照预设线路生成目标化合物的“细胞工厂”具有成本低、耗时短、绿色环保等优势, 而获得高效的基因元件是异源合成的基础。
当前, 雷公藤红素和雷公藤内酯甲生物合成途径均已基本解析, 其生物合成路线如图1所示。对于雷公藤红素, 首先前体2,3-氧化鲨烯 (2,3-oxidosaqualene) 经氧化鲨烯环化酶TwOSC1/3催化生成木栓烷型三萜骨架木栓酮 (friedelin) [11]; 随后TwCYP712K1/2/3/5能催化木栓酮C-29位羧化生成美登木酸 (polpunonic acid)[12,13]; 而TwCYP716C52和TwCYP81AM1/2则分别催化美登木酸C-2位羟化和C-24位羧化, 最终形成雷公藤红素酸 (celastrogenic acid), 该化合物经过简单的化学转化即能产生雷公藤红素[14]。对于雷公藤内酯甲, 首先前体2,3-氧化鲨烯在TwOSC1/2/3/8催化下形成齐墩果烷型三萜骨架β-香树素 (β-amyrin); 随后多功能酶TwCYP712K1/2/3/5催化β-香树素C-29位羧化生成3-表卡通酸 (3-epi-katonic acid); 后者经TwCYP82AS1发生C-22位羟化形成雷公藤三萜酸A (abrusgenic acid), 最后该化合物在对甲苯磺酸 (TsOH) 的催化下发生酯化反应生成雷公藤内酯甲[13]。
近年来, 通过定向突变技术改造药用植物活性成分生物合成途径中蛋白的催化功能已成为该领域的研究热点, 取得了显著的研究成果。如Sun等[15]通过计算与实验相结合的方法确定了影响CYP72A63与底物β-香树素结合的关键氨基酸残基, 通过构建不同突变体使其选择性氧化为羟化或羧化产物, 从而控制了酶的化学选择性和区域选择性; Xu等[16]通过将倍半萜合酶SvES与已知结构的倍半萜合酶SdS进行序列比对, 确定了定点突变的氨基酸残基并将其突变成结构更小的氨基酸, 从而拓宽酶的活性中心空腔, 获得的两个关键突变体F77I和F77A可分别使SvES转变为二萜或二倍半萜合酶; Grobe等[17]通过改造CYP107D1, 获得了几乎具有完全区域和立体选择性的三重突变体 (F84Q/S240A/V291G), 使其专一催化石胆酸 (litho chalic acid, LCA) 的7β位发生羟化生成熊去氧胆酸 (ursodeoxycholic acid, UDCA), 较S240A单突变体产量高10倍。因此通过定向突变, 获得关键的氨基酸位点, 对于改善或提高CYP450的催化功能具有重要意义。
TwCYP712K1/2/3/5是雷公藤红素和雷公藤内酯甲生源合成途径中的首个CYP450基因, 深入探究其催化机制有助于获得活性改善的CYP450蛋白。课题组前期研究发现, TwCYP712K1/2/3/5虽均能催化木栓酮和β-香树素C-29位羧化生成相应的产物, 但催化活性和底物亲和力并不相同, 此外, 在进化上四条TwCYP712Ks属于同源基因, 序列相似度较高。因此, 本研究拟通过对四条TwCYP712Ks进行同源建模并模拟蛋白与底物空间结构, 比较位于四条蛋白活性口袋内的氨基酸残基, 选取可能影响催化功能的差异氨基酸进行互突或突变成极性相同或相反的氨基酸, 探究活性口袋周围氨基酸对蛋白催化功能的影响。通过定点突变实验, 明确影响TwCYP712Ks催化活性和底物选择性的关键氨基酸, 获得高效优势的突变体元件, 进而用于活性三萜化合物的合成生物学生产。
材料与方法
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菌株、质粒和载体   大肠杆菌Trans1-T1感受态细胞购自北京全式金生物技术有限公司; BY-T3酵母菌 (BY4742-△TRP, δDNA: PPGK1-tHMG1-TADH1-PTEF1-LYS2-TCYC1, TRP1::HIS3-PPGK1-ERG20-TADH2-PTDH3-ERG1-TTPI1-PTEF1-ERG9-TCYC1) 由中国科学院天津工业生物技术研究所戴住波课题组惠赠; pEASY-Blunt Zero::-TwCYP712K1/2/3/5、pESC-Leu::(TwOSC1T502Eopt+TwCPR3opt)、pYES2-Ura::GgbAS1、pESC-Ura::gRNA、pESC-Trp::cas9质粒以及p416 GPD真核表达载体均为实验室前期构建或保存。
试剂   Gene JET Gel Extraction Kit (Thermo Scientific公司); 快速质粒小提试剂盒 (北京天根生化科技有限公司); 酵母基因组DNA提取试剂盒 (北京索莱宝科技有限公司); BamHI-HF®和SalI-HF®限制性内切酶 (美国NEB公司); 2×Phanta® Flash Master Mix、2×Rapid Taq Master Mix和CloneExpress® Ultra One Step Cloning Kit (南京诺唯赞生物科技有限公司); Fast Mutagenesis System (北京全式金生物技术有限公司); 酵母选择营养缺陷培养基SD-Trp-His-Ura和SD-Trp-His (北京泛基诺科技有限公司); β-香树素 (纯度98%)、木栓酮 (纯度98%)、3-表卡通酸 (纯度98%) 及美登木酸 (纯度98%) 均购自上海源叶生物科技有限公司; 引物合成及测序 (北京睿博兴科生物科技有限公司)。
底盘菌构建与验证    采用CRISPR-Cas9基因编辑技术[18]将TwOSC1T502Eopt、TwCPR3opt和GgbAS1表达盒整合至酿酒酵母BY-T3菌株染色体X-2位点。首先, 通过重叠延伸PCR克隆得到3个模块: ① X2up-TDH3p-TwOSC1T502Eopt-ADH1t-tHXT7p; ② tHXT7p-GgbAS1-FBA1t-PGK1p; ③ PGK1p-TwCPR3opt-TDH2t-X2down, 引物如表1所示。随后, 将这些线性DNA片段与pESC-Ura::gRNA和pESC-Trp::cas9质粒共同转化至BY-T3感受态细胞。最后, 使用5-氟乳清酸 (5-FOA) 清除带有URA3筛选标记的gRNA质粒。按照酵母基因组DNA提取试剂盒说明书提取酵母基因组, 设计引物PCR1～4-F/R (表1), 采用2× Rapid Taq Master Mix进行PCR反应, 鉴定各模块是否成功整合至BY-T3菌株相应位置。阳性菌株经测序, 命名为BY-SH。
BY-SH在SD-Trp-His (培养基均含2%葡萄糖) 固体平板划线, 30 ℃培养2天后, 挑取3个单克隆菌落于3 mL SD-Trp-His液体培养基中活化, 待菌液浑浊后, 以初始A600为0.05接种于20 mL SD-Trp-His培养基中, 培养4天。离心收集菌体, 加入10 mL 20% KOH和50% EtOH溶液, 加热回流5 min提取产物, 上清液用等体积的正己烷萃取3次。合并萃取液, 旋蒸蒸干溶剂后, 加入100 μL的无水吡啶和100 μL三氟乙酰胺 (含1%三甲基硅烷) 65 °C衍生化1 h。衍生化完成后, 氮吹吹干衍生化试剂。用200 μL乙酸乙酯复溶产物, 过0.22 μm微孔滤膜。使用美国Agilent 7890B气相色谱串联7000C GC/MS质谱检测产物, 色谱柱为DB-5MS (15 m × 0.25 mm, 0.10 μm); 柱温箱起始温度设置为50 ℃, 保持1 min, 以50 ℃·min-1梯度升温至270 ℃, 后以20 ℃·min-1升温至305 ℃, 然后保持12 min; 离子源温度设为250 ℃, 电离能70 eV, 质量扫描范围设为m/z 10～550; 进样量1 μL。
TwCYP712Ks真核表达载体构建   将TwCYP712K1/2/3/5构建至低拷贝真核表达载体p416 GPD-Ura, 以实验室前期构建的pEASY-Blunt Zero::TwCYP712K1/2/3/5为模板质粒, 设计引物 (表1) 扩增带有同源臂的目的基因片段, 反应体系为: 2×Phanta® Flash Master Mix 25 μL, 正反向引物各2.5 μL, 模板质粒1 μL, ddH2O 19 μL。反应程序: 98 ℃ 30 s; 98 ℃ 10 s, 60 ℃ 10 s, 72 ℃ 1 min, 35个循环; 72 ℃ 5 min; 4 ℃保持, 经电泳检测后切胶回收PCR产物; 使用BamHI-HF®和SalI-HF®对p416 GPD-Ura表达载体进行双酶切, 获得线性载体片段; 采用CloneExpress® Ultra One Step Cloning Kit试剂盒对目的片段和载体进行重组连接, 50 ℃反应25 min。将重组质粒p416::TwCYP712K1/2/3/5转化至Trans1-T1感受态细胞中, 涂布于含氨苄青霉素的LB固体培养基上, 于37 ℃倒置过夜培养, 挑选单菌落进行菌落PCR验证, 并将阳性结果送测序验证, 选取测序结果正确的菌液保菌并提取质粒。
转化酵母和发酵   新鲜活化的BY-SH按照A600为0.05接种到20 mL SD-Trp-His液体培养基中, 培养至A600达到0.8～1.0, 采用醋酸锂转化法, 制备BY-SH酵母感受态; 将对照质粒p416 GPD和p416::TwCYP712K1/2/3/5重组质粒转化至感受态中, 配制反应体系: 50% PEG 3350 120 μL, 1 mol·L-1 LiAc 18 μL, ssDNA 5 μL、质粒2 μL, ddH2O 35 μL。于30 ℃培养箱内放置30 min; 随后42 ℃热激15 min; 3 600 r·min-1, 离心5 min后收集菌体, 在超净台内吸去上层转化体系, 并用100 μL无菌水重悬后涂布于SD-Trp-His-Ura固体培养基上, 30 ℃培养3天后, 从每个平板上分别挑取3个单菌落至3 mL SD-Trp-His-Ura液体培养基中, 培养至菌液混浊后, 按照A600 = 0.1接入5 mL YPD培养基中, 30 ℃培养4天。
产物提取和检测   在发酵液中加入等体积乙酸乙酯超声提取两次, 合并有机相, 采用旋转蒸发仪蒸干溶剂后, 加入100 μL色谱甲醇复溶。高效液相色谱-高分辨质谱 (Q Exactive HF, 美国Thermo Scientific公司) 定量检测产物, 流动相A相为0.1%甲酸-水溶液, B相为纯乙腈溶液, 使用ACQUITY UPLC HSS T3色谱柱 (100 mm × 2.1 mm, 1.8 μm) 进行分离, 流速0.4 mL·min-1, 进样量5 μL, 柱温设置为40 ℃, 液相梯度程序为0～4 min, 30%～83% B; 4～6 min, 83% B; 6～8 min, 83%～85% B; 8～10 min, 85% B; 10～13 min, 85%～100% B; 13～15 min, 100%～30% B。使用正离子模式下的分段扫描方法进行检测, 质谱检测条件为质量范围m/z 450～460, 扫描时间0.2 s, 鞘气流速45 L·min-1, 毛细管温度320 ℃, 电喷雾电流0.4 µA, 检测结果用Xcalibur软件进行分析。将定量数据在Graphpad Prism 10软件上进行独立样本t检验, 分析统计样本间的差异性。
蛋白序列比对分析   采用Clustal X (http://www.clustal.org/clustal2/) 和ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/) 在线网站对四条TwCYP712Ks的氨基酸序列进行多重序列比对, 并使用MEME在线网站 (https://memesuite.org/meme/tools/meme) 对TwCYP712Ks进行保守基序分析。
蛋白建模和分子对接   采用I-TASSER (https://seq2fun.dcmb.med.umich.edu//I-TASSER/) 网站分别对TwCYP712K1/2/3/5蛋白进行从头建模, 并采用ProSA (https://prosa.services.came.sbg.ac.at/prosa.php/) 检测蛋白质氨基酸残基间相互作用能的合理性。从Pubchem (https://pubchem.ncbi.nlm.nih.gov/) 官方网站下载小分子化合物木栓酮 (Pubchem CID: 91472) 和β-香树素 (Pubchem CID: 73145) 的三维结构, 并用Open Bable 软件将sdf格式转化为pdb格式。
使用Autodock 4.2.6软件对TwCYP712K1/2/3/5蛋白进行加氢、加电荷处理, 原子类型选择为Assign AD4 type, 并保存为pdbqt格式。对小分子木栓酮和β-香树素进行加氢和加电荷处理, 保存为pdbqt格式。通过TwCYP712K1/2/3/5蛋白中亚铁血红素 (HEM) 的位置, 确定对接区域中心, 调整对接区域大小, 使HEM被完全包裹, 保存对接Grid box。设置对接参数后, 运行Autodock进行分子对接, 得到对接结果文件dlg。使用AutoDockTools软件输出小分子和蛋白复合结构, 随后用PyMOL软件进行可视化处理。
定点突变   分析TwCYP712K1/2/3/5蛋白与底物木栓酮和β-香树素的对接结果, 选取活性口袋附近的氨基酸作为研究对象, 结合多重序列比对及蛋白保守基序分析结果, 基于差异氨基酸互相突变的策略, 研究活性口袋及周围氨基酸的作用。按照Fast Mutagenesis System试剂盒说明书, 利用SnapGene软件设计点突变引物, 以p416::TwCYP712K1/2/3/5为模板质粒, 进行突变载体扩增。反应体系: 模板质粒10 ng, 2×TransStart® Fastpfu PCR Super Mix 10 μL, 正反向引物各1 μL, ddH2O补足至20 μL。反应程序: 94 ℃ 3 min, 94 ℃ 20 s, 56 ℃ 20 s, 72 ℃ 4 min, 25个循环; 72 ℃ 10 min; 4 ℃保持。经1.0% 琼脂糖凝胶电泳分离确定目的条带位置正确的样品, 加入0.5 μL DMT酶, 充分混匀后37 ℃恒温孵育1 h, 消除甲基化质粒模板。取2～5 μL消化产物转化至50 μL DMT感受态细胞中, 涂布于含氨苄青霉素的 LB 固体培养基上, 于37 ℃过夜培养。将测序验证正确的单克隆菌液保菌, 提取质粒, 得到TwCYP712Ks突变体。将p416::TwCYP712K1/2/3/5野生型 (wild type, WT) 和突变体分别转化至BY-SH感受态细胞中, 转化、发酵及产物提取检测方法如前所示。
结果与分析
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1 自生产β-香树素和木栓酮的底盘菌构建
采用GC-MS检测含有TwOSC1T502Eopt、GgbAS1和TwCPR3opt片段的酵母菌株BY-SH的发酵产物 (图2B), 结果显示, 与对照组相比, 在总离子流图 (TIC) 模式下, 该菌株能产生两个新峰 (1和2)。通过检索质谱数据库并与对照品β-香树素和木栓酮的保留时间和质谱碎片信息比对 (图2C), 确定峰1为β-香树素, 峰2为木栓酮, 证明TwOSC1T502Eopt、TwCPR3opt和GgbAS1基因已成功整合至原菌株BY-T3基因组上, 如图2A所示。
2 TwCYP712Ks催化活性分析
将TwCYP712K1/2/3/5基因构建至表达载体p416 GPD, 将对照质粒及重组质粒分别转入BY-SH酵母菌株, 使用高分辨质谱检测发酵产物。提取β-香树素和木栓酮的羧化产物[M+H]+, m/z 457.368 0。如图2E显示, TwCYP712K1/2/3/5与对照组相比, 均出现两个新的产物峰 (3和4)。通过与标准品保留时间和质谱碎片信息 (图2F、G) 对比, 确定两个羧化产物分别为3-表卡通酸 (3) 和美登木酸 (4)。
四个TwCYP712Ks酶均能催化木栓酮和β-香树素分别生成美登木酸和3-表卡通酸, 但催化活性有区别。其中TwCYP712K1主要催化木栓酮羧化产生美登木酸, 少量生成3-表卡通酸, 而TwCYP712K2/3则主要催化β-香树素羧化产生3-表卡通酸, 而美登木酸产量相对较低, TwCYP712K5催化生成的美登木酸和3-表卡通酸产量相当, 前者略高于后者。通过定量发酵检测产物3和4, 结果如图2D显示, 催化木栓酮生成美登木酸活性最强的是TwCYP712K1, 其生成美登木酸的产量约是活性最弱的TwCYP712K3的39倍; 催化β-香树素生成3-表卡通酸活性最强的是TwCYP712K2, 其生成3-表卡通酸的产量约是活性最弱的TwCYP712K1的24倍; TwCYP712K5对两种底物的催化活性均较弱。
3 TwCYP712Ks蛋白序列比对分析
多重序列比对结果显示 (图3A), 四条TwCYP712Ks亲缘关系较近, 序列相似度较高, 总体相似度为85.44%, 两两氨基酸序列相似度如表2所示。采用MEME软件预测了TwCYP712Ks蛋白的10个motif (图3B), 其中研究相对较多的保守基序FxxGxRxCxG和ExLR分别包含在motif 1和motif 8中, 研究表明FxxGxRxCxG结构域含高度保守的半胱氨酸 (Cys), 是血红素的结合区域, 而ExLR结构域具有稳定核心结构的作用[19]。
4 TwCYP712Ks蛋白建模与对接分析
对于蛋白质改造和分子对接, 有助于理解反应催化机制并获得活性更优的突变体。采用ProSA 检测蛋白三维结构氨基酸残基之间的相互作用能, 结果表明计算的Z值 (TwCYP712K1/2/3/5分别为-7.66、-7.35、-7.53和-7.91) 均为负值且位于ProSA能量图范围内, 证明4条建模蛋白结构的能量均具有合理性。
模拟TwCYP712K1/2/3/5与底物木栓酮和β-香树素对接的三维空间结构, 结果显示, 两个底物的C-29位均指向亚铁血红素 (HEM) 活性中心, 选取位于活性口袋中距离底物分子5 Å范围内的氨基酸作为研究对象, 如图4所示。将TwCYP712K1分别与TwCYP712K2、TwCYP712K3以及TwCYP712K5的对接结果进行比对, 选取空间位置相同但氨基酸不同的残基进行互突或突变成极性相同或相反的氨基酸, 来研究活性口袋周围氨基酸的作用, 共产生63个突变方向。
5 TwCYP712Ks突变结果分析
为了探究各突变体对TwCYP712Ks功能的影响, 将构建成功的突变表达载体和野生型p416::TwCYP712K1/2/3/5分别转入BY-SH中发酵, 提取产物进行检测。结果显示 (图5A), TwCYP712K1表现为主要催化木栓酮产生美登木酸, 大部分突变体与野生型产量相当或有不同程度的降低, 其中以I125F和V379L下降最为明显, 分别是野生型产量的24%和26%, 说明125位的异亮氨酸 (I) 和379位的缬氨酸 (V) 对于TwCYP712K1催化木栓酮C-29位羧化生成美登木酸影响较大, 且突变后的苯丙氨酸 (F) 和亮氨酸 (L) 相对于I和V氨基酸空间位阻均增大了, 可能使底物与蛋白的结合空间减小, 导致TwCYP712K1催化活性减弱; 少数突变位点使美登木酸产量提升, 其中最显著的是T488A和P378S, 分别为野生型的1.43和1.42倍; TwCYP712K1催化生成3-表卡通酸产量远低于美登木酸, 且突变体与野生型产量差异无统计学意义。
对于主产3-表卡通酸的TwCYP712K2, 其突变体功能与野生型相比差异较大 (图5B), 一些位点突变之后甚至导致TwCYP712K2产物比例发生逆转, 使美登木酸产量高于3-表卡通酸。首先, I126V、F127I、A227T及A490T突变体对美登木酸产量提高最为明显, 分别是野生型的2.49、5.19、6.26和2.86倍, 此外A490T也使3-表卡通酸产量提高了2.18倍, 这些氨基酸相对于底物木栓酮或β-香树素的空间位置如图5B所示。为了探究这些位点组合突变后产量是否会有进一步提升, 分别构建了双突变I126V-F127I和三突变I126V-F127I-A227T载体, 结果与预期一致, 联合突变使美登木酸产量分别提高了8.00和12.39倍, 说明V126、I127和T227决定TwCYP712Ks催化木栓酮C-29位的高催化活性。此外, F127I与A227T突变体会导致3-表卡通酸产量下降, 说明127位的苯丙氨酸 (F) 和227位的丙氨酸 (A) 是影响TwCYP712K2与底物结合的关键氨基酸残基, 突变之后, 导致蛋白从原来的倾向于与β-香树素结合转变为易于与木栓酮结合, 从而使美登木酸产量高于3-表卡通酸。值得注意的是, 一些氨基酸位点突变之后导致美登木酸和3-表卡通酸产量均降低了, 尤其以F445D和L226M变化最为明显, 分别使美登木酸产量降低了89%和93%, 而3-表卡通酸几乎检测不到。
对于TwCYP712K3, 虽然主产物也是3-表卡通酸, 但其活性弱于TwCYP712K2, 且二者氨基酸序列相似度高达90.27%, 故本研究选择了3个位点突变体验证TwCYP712K2上述结果。其中, I127V突变体对TwCYP712K3活性影响较为明显, 使3-表卡通酸产量提高了2.53倍, 推测异亮氨酸 (I) 突变成结构更小的缬氨酸 (V) 后拓宽了酶的活性中心空腔, 使TwCYP712K3更易于与底物结合 (图5C); 此外, 构建的双突载体I127V-F128I则会导致3-表卡通酸产量降低, 说明TwCYP712K2的其他氨基酸对整体功能的影响较大。
对于TwCYP712K5 (图5D), 在选择的5个突变方向中, K114E、S378P及F491L均使3-表卡通酸产量有所提高, 分别是野生型的2.82、2.04和1.41倍, 且突变方向均为TwCYP712K2相应位置的氨基酸, 说明这几个位点对于提高蛋白与底物β-香树素的亲和性有重要意义; T110V和S378P对美登木酸产量提升较为明显, 分别是野生型的1.92倍和2.97倍, 推测将极性苏氨酸 (T) 突变成非极性缬氨酸 (V) 之后, 氨基酸疏水性显著提高, 有利于蛋白与底物结合; 而脯氨酸 (P) 为TwCYP712K1相应位置的氨基酸, 其突变之后美登木酸产量也有所提高。此外, 根据TwCYP712K2的双突I126V-F127I结果, 也分别构建了其他三条基因相应位置的双突突变体, 结果发现TwCYP712K1/3产量变化不明显, 而对TwCYP712K5蛋白活性影响较大。当发酵检测TwCYP712K5的I125V-L126I突变体时, 发现美登木酸产量只有野生型的17%, 而3-表卡通酸几乎检测不到, 推测这两个位点对TwCYP712K5功能有重要影响。
讨论
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雷公藤中三萜化合物雷公藤红素和雷公藤内酯甲具有广泛的药理活性, 随着越来越多活性物质作用的信号通路和靶点被逐渐揭示, 二者及其衍生物有极大的可能性被开发成药物用于相关疾病的治疗。近年来, 基于对药用天然产物生物合成途径和转录调控的研究, 以及多组学、生物信息学、分子生物学等相关领域基础理论及技术的发展, 植物生物反应器和微生物细胞工厂正在成为合成所需天然产物的替代手段[20]。然而植物来源的基因元件在异源宿主中的表达和催化功能常受到限制, 存在CYP450对底物的特异性催化机制不明确、异源宿主中表达率低或与氧化还原伴侣CPR适配性差等问题, 限制了目标化合物的异源高效生物合成。
定向进化是一种利用人工手段对目的蛋白进行改造的技术, 属于蛋白质的非理性设计, 其优势在于能在很短的时间内积累自然界上万年才能产生的变异, 从而对蛋白质的活性、底物普适性、热稳定性、催化反应类型等方面进行特异性改造[21]。近年来, 定向进化技术已成为改造CYP450酶[15]、萜类合酶[16]、糖基转移酶[22]等生物合成途径中蛋白元件的有效手段。
本研究以雷公藤红素和雷公藤内酯甲生源途径上的第一个CYP450酶TwCYP712K1/2/3/5为研究对象, 对其进行蛋白建模、分子对接和定点突变实验, 共构建了63个突变体元件, 筛选到了一系列影响酶活性的关键氨基酸残基, 为C-29位羧化机制的解析奠定了基础。尤其是通过同源基因多重序列比对及蛋白活性口袋比较, 利用差异氨基酸互相突变的方式研究蛋白催化活性的变化, 可高效发掘影响活性的氨基酸残基, 揭示了F/I127、A/T227位点是决定TwCYP712Ks与底物β-香树素或木栓酮亲和的关键氨基酸残基。
TwCYP712K1/2/3/5催化木栓酮和β-香树素生成美登木酸和3-表卡通酸实际上经历了三步催化反应 (羟化、醛基化、羧化), 受限于检测条件和缺乏中间体对照品, 本研究仅关注了羧基化终产物。因此后续研究可通过优化检测方法, 对产物进行富集、制备及核磁鉴定, 深入探究TwCYP712Ks的催化机制, 明确决定羟、醛、酸产物的关键氨基酸残基, 全面解析TwCYP712Ks的底物和化学选择性。
作者贡献: 刘攀婷进行了本研究的实验操作、数据分析和论文初稿撰写; 张逸风设计了实验方法、数据分析和论文撰写; 刘远、高杰、马林、胡雅婷、苏平和袁仕君提供了实验技术支持; 吴晓毅分析了相关实验数据; 高伟和张夏楠构思了实验方案和论文修改。
利益冲突: 所有作者均声明不存在利益冲突。
基金
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  • 国家自然科学基金面上项目(81973418)
  • 国家自然科学基金面上项目(81974515)
  • 国家重点研发计划项目(2020YFA0908000)
  • 国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005)
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2024年第59卷第6期
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doi: 10.16438/j.0513-4870.2023-1325
  • 接收时间：2023-11-24
  • 首发时间：2025-11-26
  • 出版时间：2024-06-12
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  • 收稿日期：2023-11-24
  • 修回日期：2024-01-16
基金
国家自然科学基金面上项目(81973418)
国家自然科学基金面上项目(81974515)
国家重点研发计划项目(2020YFA0908000)
国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005)
作者信息
    1.首都医科大学中医药学院, 北京 100069
    2.道地药材品质保障与资源持续利用全国重点实验室, 中国中医科学院中药资源中心, 北京 100700

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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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