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Article(id=1241451295949771099, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241451293068284204, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240025, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1704643200000, receivedDateStr=2024-01-08, revisedDate=null, revisedDateStr=null, acceptedDate=1709740800000, acceptedDateStr=2024-03-07, onlineDate=1773914654003, onlineDateStr=2026-03-19, pubDate=1710172800000, pubDateStr=2024-03-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773914654003, onlineIssueDateStr=2026-03-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773914654003, creator=13701087609, updateTime=1773914654003, updator=13701087609, issue=Issue{id=1241451293068284204, tenantId=1146029695717560320, journalId=1192105938417971205, year='2024', volume='64', issue='8', pageStart='2591', pageEnd='3085', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773914653317, creator=13701087609, updateTime=1773919071204, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241469823079731774, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241451293068284204, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241469823079731775, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241451293068284204, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2752, endPage=2767, ext={EN=ArticleExt(id=1241451297719767419, articleId=1241451295949771099, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Efficient synthesis of ergothioneine in Actinoplanes sp., columnId=1241045257748533520, journalTitle=Acta Microbiologica Sinica, columnName=Research Articles, runingTitle=null, highlight=null, articleAbstract=

[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.

, correspAuthors=Tiangang LIU, authorNote=null, correspAuthorsNote=
*LIU Tiangang, E-mail:
, copyrightStatement=Copyright ©2024 Acta Microbiologica 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=Zhen LIU, Yingying CHANG, Zixin DENG, Tiangang LIU), CN=ArticleExt(id=1241451299267465704, articleId=1241451295949771099, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=麦角硫因在游动放线菌底盘中的高效合成, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

【目的】麦角硫因是一种稀有的天然氨基酸类强抗氧化剂，在机体内发挥着重要的生理功能，并在食品、医药、化妆品领域得到广泛应用。然而，传统的从蕈菇中提取和化学合成的方法存在收率低、成本高等问题，本研究期望利用代谢工程改造，提高游动放线菌(Actinoplanes sp.) HS中麦角硫因的产量。【方法】首先通过生物信息学分析，锁定了Actinoplanes sp. HS中可能参与麦角硫因合成的基因；接着在大肠杆菌(Escherichia coli) BL21(DE3)中进行异源表达，进一步鉴定了这些基因的功能；最后将鉴定功能的基因组合后在Actinoplanes sp. HS中进行高表达，对突变株产量进行测定，并通过在发酵培养基中添加不同浓度的前体，探究其对发酵产量的影响。【结果】Actinoplanes sp. HS中基因BC03-04016、BC03-04015、BC03-04014、BC03-04013所编码的酶可以合成麦角硫因前体组氨酸甜菜碱半胱氨酸亚砜(hercynine-cysteine-sulphoxide, HER-Cys-Sul)，基因BC03-04046和BC03-04917所编码的酶都具有裂解碳硫键的功能，可以催化前体HER-Cys-Sul形成终产物麦角硫因。将编码麦角硫因合成的基因高表达后得到的突变株YC313和YC314，其麦角硫因产量分别达到125 mg/L和108 mg/L，较出发菌株Actinoplanes sp. HS分别提高至2.9倍和2.5倍。在发酵培养基中额外添加0.35 g/L的甲硫氨酸后，YC313菌株的麦角硫因产量相较于原始培养基提高了24%；额外添加10 g/L的黄豆饼粉后，相较于原始培养基麦角硫因产量提高了19%。【结论】本研究对Actinoplanes sp. HS中麦角硫因合成的相关基因及其功能进行了鉴定，通过代谢工程改造得到了两株麦角硫因高产菌株，并进一步研究了前体供应对麦角硫因产量的影响，为以游动放线菌为底盘生产麦角硫因提供了策略支持。

, correspAuthors=刘天罡, authorNote=null, correspAuthorsNote=null, copyrightStatement=版权所有©《微生物学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=IjHkwLKBWiB3xO89i855hw==, magXml=BTjruqwNz9B43LXvlGJcNg==, pdfUrl=null, pdf=8qL86bsH5UyUZIH6fur4+w==, pdfFileSize=1038477, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=lk0JNMjUa4vHGB+jH8ExqA==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=4+yMQWHwIWTPHjJsVOZFOw==, mapNumber=null, authorCompany=null, fund=null, authors=

#These authors contributed equally to this work.

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A: Schematic diagram of the structure of plasmids pCIL001A and pCIL001. B: Accurate molecular weight extraction ion chromatograms of HER-Cys-Sul., figureFileSmall=tQMhjpKihGLQM/et9ZQtnA==, figureFileBig=VV9BYGKwyby362KRY+k4ow==, tableContent=null), ArticleFig(id=1242193064119333662, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=图2, caption=HER-Cys-Sul合成基因质粒构建及异源表达菌株中产物检测, figureFileSmall=tQMhjpKihGLQM/et9ZQtnA==, figureFileBig=VV9BYGKwyby362KRY+k4ow==, tableContent=null), ArticleFig(id=1242193064257745702, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Figure 3, caption=Plasmid construction of carbon-sulfur lyase gene and detection of products in heterologous expression strains. A: Schematic diagram of the structure of plasmids pCIL003 and pCIL008. B: Accurate molecular weight extraction ion chromatogram of HER-Cys-Sul in different mutant strains fermentation broth. C: Comparison of secondary mass spectrometry fragments from fermentation broth of YC303 strain and ergothioneine standard. D: Titer of ergothioneine in different mutant strains., figureFileSmall=sSV9+usvKWdbxQZN5j6MAw==, figureFileBig=pfS6vYopEBN3hCfPp/9/Dw==, tableContent=null), ArticleFig(id=1242193064366797614, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=图3, caption=碳硫裂解酶基因质粒构建及异源表达菌株中产物检测, figureFileSmall=sSV9+usvKWdbxQZN5j6MAw==, figureFileBig=pfS6vYopEBN3hCfPp/9/Dw==, tableContent=null), ArticleFig(id=1242193064459072310, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Figure 4, caption=Construction of ergothioneine high-yield strains and verification of mutant strains. A: Schematic diagram of overexpression plasmid construction. B: PCR verification of overexpression strains YC313 and YC314 agarose gel electropherosis. C: Titer of overexpressed strains YC313 and YC314 with the original strains of ergothioneine. ***: P < 0.001., figureFileSmall=58v7g52kKEY9pxRLP8CMqA==, figureFileBig=zh14K2r2Nye9W29l9hoMRw==, tableContent=null), ArticleFig(id=1242193064580707140, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=图4, caption=麦角硫因高产菌株的构建及突变株验证, figureFileSmall=58v7g52kKEY9pxRLP8CMqA==, figureFileBig=zh14K2r2Nye9W29l9hoMRw==, tableContent=null), ArticleFig(id=1242193064693953356, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Figure 5, caption=Effect of different precursors (A) and culture media components (B) on ergothioneine titer. The original culture medium is used as the control. *: P < 0.05; **: P < 0.01; ***: P < 0.001., figureFileSmall=1LMc5RMGcHQq/ESUv+0etA==, figureFileBig=l9x6VzA1VYlkXRdA4cS2fw==, tableContent=null), ArticleFig(id=1242193064807199575, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=图5, caption=不同前体和培养基成分添加对麦角硫因产量的影响, figureFileSmall=1LMc5RMGcHQq/ESUv+0etA==, figureFileBig=l9x6VzA1VYlkXRdA4cS2fw==, tableContent=null), ArticleFig(id=1242193064962388827, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Table 1, caption=

Strains and plasmids used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
NameDescription
Strains
Actinoplanes sp. HSWild-type
ET12567-pUZ8002recF, dam, dcm, hsdS, for intergeneric conjugation
Escherichia coli BL21 StarTM (DE3)F−dcm ompT hsdS (rB−mB−) galλ (DE3)
YC300E. coli BL21(DE3) with plasmid pETDuet-1
YC301E. coli BL21(DE3) with plasmid pCIL001
YC303E. coli BL21(DE3) with plasmid pCIL003
YC308E. coli BL21(DE3) with plasmid pCIL008
YC313Integrated the plasmid pCIL013 in the genome of Actinoplanes sp. HS
YC314Integrated the plasmid pCIL014 in the genome of Actinoplanes sp. HS
Plasmids
pETDuet-1Protein expression vectors for E. coli
pBBR1MCS-2Protein expression vectors for E. coli
pSET152E. coli replicon, att, oriT, aac(3)IV
pIB139E. coli replicon, att, oriT, aac(3)IV, ermE*
pCIL001ApETDuet-1 carries BC03-04016-BC03-04015 under the control of T7 promoter
pCIL001pCIL001A carries BC03-04014-BC03-04013 under the control of T7 promoter
pCIL003pBBR1MCS-2 carries BC03-04046 under the control of T3 promoter
pCIL008pBBR1MCS-2 carries BC03-04917 under the control of T3 promoter
pCIL013pSET152 carries BC03-04016-BC03-04015-BC03-04014-BC03-04013 under the control of ermE* promoter and BC03-04046 under the control of ermE* promoter
pCIL014pSET152 carries BC03-04016-BC03-04015-BC03-04014-BC03-04013 under the control of ermE* promoter and BC03-04917 under the control of ermE* promoter
), ArticleFig(id=1242193065100800864, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=表1, caption=

本研究所用菌株和质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
NameDescription
Strains
Actinoplanes sp. HSWild-type
ET12567-pUZ8002recF, dam, dcm, hsdS, for intergeneric conjugation
Escherichia coli BL21 StarTM (DE3)F−dcm ompT hsdS (rB−mB−) galλ (DE3)
YC300E. coli BL21(DE3) with plasmid pETDuet-1
YC301E. coli BL21(DE3) with plasmid pCIL001
YC303E. coli BL21(DE3) with plasmid pCIL003
YC308E. coli BL21(DE3) with plasmid pCIL008
YC313Integrated the plasmid pCIL013 in the genome of Actinoplanes sp. HS
YC314Integrated the plasmid pCIL014 in the genome of Actinoplanes sp. HS
Plasmids
pETDuet-1Protein expression vectors for E. coli
pBBR1MCS-2Protein expression vectors for E. coli
pSET152E. coli replicon, att, oriT, aac(3)IV
pIB139E. coli replicon, att, oriT, aac(3)IV, ermE*
pCIL001ApETDuet-1 carries BC03-04016-BC03-04015 under the control of T7 promoter
pCIL001pCIL001A carries BC03-04014-BC03-04013 under the control of T7 promoter
pCIL003pBBR1MCS-2 carries BC03-04046 under the control of T3 promoter
pCIL008pBBR1MCS-2 carries BC03-04917 under the control of T3 promoter
pCIL013pSET152 carries BC03-04016-BC03-04015-BC03-04014-BC03-04013 under the control of ermE* promoter and BC03-04046 under the control of ermE* promoter
pCIL014pSET152 carries BC03-04016-BC03-04015-BC03-04014-BC03-04013 under the control of ermE* promoter and BC03-04917 under the control of ermE* promoter
), ArticleFig(id=1242193065188881256, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Table 2, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer Sequences (5′→3′)
Primer 1TTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCATGAGTTCGGCCGATCGAGTGC
Primer 2CATGGTTAATTCCTCCTTCATCGAGCGTTCCCCTTC
Primer 3TGAAGGAGGAATTAACCATGATCAACCCCGACAAGAACCTG
Primer 4CCTGCAGGCGCGCCGAGCTCGAATTCGGATCCTCAGACGAGCTCCCGCGCGCACCG
Primer 5AAGTATAAGAAGGAGATATACATATGTGCCGCCACCTGGCCTACC
Primer 6TCATAGTGTAATCCTCCTTCACAGCTCTCCTAATTCCACCG
Primer 7TGAAGGAGGATTACACTATGACTTCGCTGGAGAAGCATC
Primer 8GCAGCGGTTTCTTTACCAGACTCGAGTCAATCAGTGACGGCCTGGGCCAGGGAG
Primer 9CGCCATATGGAGCTCCAATTCGCCCTATAG
Primer 10CGCCATATGGGTTAATTCCTCCTACTGCAGG
Primer 11ACTCACTATAGGGCGAATTGGAGCTCTCACAACGGCTTGGTGACCATGGCGCTG
Primer 12TGCAGTAGGAGGAATTAACCCATATGATGGACGTCGACGCACTCCGGGCCGGCAC
Primer 13ACTCACTATAGGGCGAATTGGAGCTCCTACCACTGCCAGTGGTGCAGC
Primer 14TGCAGTAGGAGGAATTAACCCATATGATGAGCGCCGAAGATCCACC
Primer 15CGGCCAGTGCCAAGCTTGGGCTGCAGGTCGACTCTAGAGCGAGTGTCCGTTCGAGTGGC
Primer 16CGGCTCCCCGCAGCACTCGATCGGCCGAACTCATTGGATCCTACCAACCGGCACGATTG
Primer 17CTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATCCAATGAGTTCGGCCGATCGAGTGC
Primer 18TCGGGCGCAAGCCGCCACTCGAACGGACACTCGCTCAATCAGTGACGGCCTGGGCCAGG
Primer 19GGCCCAGGCCGTCACTGATTGAGCGAGTGTCCGTTCGAGTGGCGGCTTGCGCCCGATGC
Primer 20CAGCCCGGAGTGCCGGCCCGGAGTGCGTCGACGTCCATTGGATCCTACCAACCGGCACG
Primer 21GGGCACAATCGTGCCGGTTGGTAGGATCCAATGGACGTCGACGCACTCCGGGCCGGCAC
Primer 22ACAGCTATGACATGATTACGAATTCGATATCTCACAACGGCTTGGTGACCATGGCGCTG
Primer 23CGGCAATGGGCTGCGGTGGATCTTCGGCGCTCATTGGATCCTACCAACCGGCACGATTG
Primer 24TTGTGGGCACAATCGTGCCGGTTGGTAGGATCCAATGAGCGCCGAAGATCCACCGCAGC
Primer 25CAGGAAACAGCTATGACATGATTACGAATTCGATATCCTACCACTGCCAGTGGTGCAGC
Primer 26ACTCACTATAGGGCGAATTGGAGCTCTCAGCCGAACACCTTCCGCACC
Primer 27TGCAGTAGGAGGAATTAACCCATATGATGAGCTACGACGTCGCGCGGGTGCGCAAAG
Primer 28ACTCACTATAGGGCGAATTGGAGCTCTCAGAACCCGGCCTCCTTCCGGATC
Primer 29TGCAGTAGGAGGAATTAACCCATATGGTGGATTACACAGGCGATCCGCCGGTC
Primer 30GACGAATTCCTGCAGTAGGAGGAATTAACCCATATGATGGCCTACCTCGATCATGCG
Primer 31GACGAGCTCCTAACGGGGCGTCTTCCAGGACCCCGCCCGTCG
Primer 32ACTCACTATAGGGCGAATTGGAGCTCTCAGGAGCGAACTGTGTCCGCCAG
Primer 33TGCAGTAGGAGGAATTAACCCATATGATGTCGGCTCTCGACATCGTCGGCGACG
), ArticleFig(id=1242193065260184430, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=表2, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer Sequences (5′→3′)
Primer 1TTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCATGAGTTCGGCCGATCGAGTGC
Primer 2CATGGTTAATTCCTCCTTCATCGAGCGTTCCCCTTC
Primer 3TGAAGGAGGAATTAACCATGATCAACCCCGACAAGAACCTG
Primer 4CCTGCAGGCGCGCCGAGCTCGAATTCGGATCCTCAGACGAGCTCCCGCGCGCACCG
Primer 5AAGTATAAGAAGGAGATATACATATGTGCCGCCACCTGGCCTACC
Primer 6TCATAGTGTAATCCTCCTTCACAGCTCTCCTAATTCCACCG
Primer 7TGAAGGAGGATTACACTATGACTTCGCTGGAGAAGCATC
Primer 8GCAGCGGTTTCTTTACCAGACTCGAGTCAATCAGTGACGGCCTGGGCCAGGGAG
Primer 9CGCCATATGGAGCTCCAATTCGCCCTATAG
Primer 10CGCCATATGGGTTAATTCCTCCTACTGCAGG
Primer 11ACTCACTATAGGGCGAATTGGAGCTCTCACAACGGCTTGGTGACCATGGCGCTG
Primer 12TGCAGTAGGAGGAATTAACCCATATGATGGACGTCGACGCACTCCGGGCCGGCAC
Primer 13ACTCACTATAGGGCGAATTGGAGCTCCTACCACTGCCAGTGGTGCAGC
Primer 14TGCAGTAGGAGGAATTAACCCATATGATGAGCGCCGAAGATCCACC
Primer 15CGGCCAGTGCCAAGCTTGGGCTGCAGGTCGACTCTAGAGCGAGTGTCCGTTCGAGTGGC
Primer 16CGGCTCCCCGCAGCACTCGATCGGCCGAACTCATTGGATCCTACCAACCGGCACGATTG
Primer 17CTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATCCAATGAGTTCGGCCGATCGAGTGC
Primer 18TCGGGCGCAAGCCGCCACTCGAACGGACACTCGCTCAATCAGTGACGGCCTGGGCCAGG
Primer 19GGCCCAGGCCGTCACTGATTGAGCGAGTGTCCGTTCGAGTGGCGGCTTGCGCCCGATGC
Primer 20CAGCCCGGAGTGCCGGCCCGGAGTGCGTCGACGTCCATTGGATCCTACCAACCGGCACG
Primer 21GGGCACAATCGTGCCGGTTGGTAGGATCCAATGGACGTCGACGCACTCCGGGCCGGCAC
Primer 22ACAGCTATGACATGATTACGAATTCGATATCTCACAACGGCTTGGTGACCATGGCGCTG
Primer 23CGGCAATGGGCTGCGGTGGATCTTCGGCGCTCATTGGATCCTACCAACCGGCACGATTG
Primer 24TTGTGGGCACAATCGTGCCGGTTGGTAGGATCCAATGAGCGCCGAAGATCCACCGCAGC
Primer 25CAGGAAACAGCTATGACATGATTACGAATTCGATATCCTACCACTGCCAGTGGTGCAGC
Primer 26ACTCACTATAGGGCGAATTGGAGCTCTCAGCCGAACACCTTCCGCACC
Primer 27TGCAGTAGGAGGAATTAACCCATATGATGAGCTACGACGTCGCGCGGGTGCGCAAAG
Primer 28ACTCACTATAGGGCGAATTGGAGCTCTCAGAACCCGGCCTCCTTCCGGATC
Primer 29TGCAGTAGGAGGAATTAACCCATATGGTGGATTACACAGGCGATCCGCCGGTC
Primer 30GACGAATTCCTGCAGTAGGAGGAATTAACCCATATGATGGCCTACCTCGATCATGCG
Primer 31GACGAGCTCCTAACGGGGCGTCTTCCAGGACCCCGCCCGTCG
Primer 32ACTCACTATAGGGCGAATTGGAGCTCTCAGGAGCGAACTGTGTCCGCCAG
Primer 33TGCAGTAGGAGGAATTAACCCATATGATGTCGGCTCTCGACATCGTCGGCGACG
), ArticleFig(id=1242193065348264821, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=EN, label=Table 3, caption=

Gene alignment in Actinoplanes sp. HS

, figureFileSmall=null, figureFileBig=null, tableContent=
Query IDSubject IDSubject descriptionIdentity (%)
egtABC03-04016Ergothioneine biosynthesis glutamate-cysteine ligase EgtA40.45
egtBBC03-04015Ergothioneine biosynthesis protein EgtB54.99
egtCBC03-04014Ergothioneine biosynthesis protein EgtC43.15
egtDBC03-04013l-histidine N(alpha)-methyltransferase60.44
egtEBC03-04046Aminotransferase class V-fold PLP-dependent enzyme30.87
BC03-00348Cysteine desulfurase25.65
BC03-00103Aminotransferase class V-fold PLP-dependent enzyme46.15
BC03-05049Cysteine desulfurase29.73
BC03-00781Aminotransferase class V-fold PLP-dependent enzyme27.55
BC03-04917Aminotransferase class V-fold PLP-dependent enzyme27.23
), ArticleFig(id=1242193065440539518, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241451295949771099, language=CN, label=表3, caption=

Actinoplanes sp. HS中基因比对

, figureFileSmall=null, figureFileBig=null, tableContent=
Query IDSubject IDSubject descriptionIdentity (%)
egtABC03-04016Ergothioneine biosynthesis glutamate-cysteine ligase EgtA40.45
egtBBC03-04015Ergothioneine biosynthesis protein EgtB54.99
egtCBC03-04014Ergothioneine biosynthesis protein EgtC43.15
egtDBC03-04013l-histidine N(alpha)-methyltransferase60.44
egtEBC03-04046Aminotransferase class V-fold PLP-dependent enzyme30.87
BC03-00348Cysteine desulfurase25.65
BC03-00103Aminotransferase class V-fold PLP-dependent enzyme46.15
BC03-05049Cysteine desulfurase29.73
BC03-00781Aminotransferase class V-fold PLP-dependent enzyme27.55
BC03-04917Aminotransferase class V-fold PLP-dependent enzyme27.23
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麦角硫因在游动放线菌底盘中的高效合成
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刘震 1, # , 常莹莹 1, # , 邓子新 2 , 刘天罡 1, 2, *
微生物学报 | 研究报告 2024,64(8): 2752-2767
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微生物学报 | 研究报告 2024, 64(8): 2752-2767
麦角硫因在游动放线菌底盘中的高效合成
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刘震1, #, 常莹莹1, #, 邓子新2, 刘天罡1, 2, *
作者信息
  • 1 武汉大学药学院 组合生物合成与新药发现教育部重点实验室, 湖北 武汉 430071
  • 2 上海交通大学生命科学技术学院 微生物代谢国家重点实验室, 上海 200240
Efficient synthesis of ergothioneine in Actinoplanes sp.
Zhen LIU1, #, Yingying CHANG1, #, Zixin DENG2, Tiangang LIU1, 2, *
Affiliations
  • 1 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, Hubei, China
  • 2 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
出版时间: 2024-03-12 doi: 10.13343/j.cnki.wsxb.20240025
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摘要
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【目的】麦角硫因是一种稀有的天然氨基酸类强抗氧化剂，在机体内发挥着重要的生理功能，并在食品、医药、化妆品领域得到广泛应用。然而，传统的从蕈菇中提取和化学合成的方法存在收率低、成本高等问题，本研究期望利用代谢工程改造，提高游动放线菌(Actinoplanes sp.) HS中麦角硫因的产量。【方法】首先通过生物信息学分析，锁定了Actinoplanes sp. HS中可能参与麦角硫因合成的基因；接着在大肠杆菌(Escherichia coli) BL21(DE3)中进行异源表达，进一步鉴定了这些基因的功能；最后将鉴定功能的基因组合后在Actinoplanes sp. HS中进行高表达，对突变株产量进行测定，并通过在发酵培养基中添加不同浓度的前体，探究其对发酵产量的影响。【结果】Actinoplanes sp. HS中基因BC03-04016、BC03-04015、BC03-04014、BC03-04013所编码的酶可以合成麦角硫因前体组氨酸甜菜碱半胱氨酸亚砜(hercynine-cysteine-sulphoxide, HER-Cys-Sul)，基因BC03-04046和BC03-04917所编码的酶都具有裂解碳硫键的功能，可以催化前体HER-Cys-Sul形成终产物麦角硫因。将编码麦角硫因合成的基因高表达后得到的突变株YC313和YC314，其麦角硫因产量分别达到125 mg/L和108 mg/L，较出发菌株Actinoplanes sp. HS分别提高至2.9倍和2.5倍。在发酵培养基中额外添加0.35 g/L的甲硫氨酸后，YC313菌株的麦角硫因产量相较于原始培养基提高了24%；额外添加10 g/L的黄豆饼粉后，相较于原始培养基麦角硫因产量提高了19%。【结论】本研究对Actinoplanes sp. HS中麦角硫因合成的相关基因及其功能进行了鉴定，通过代谢工程改造得到了两株麦角硫因高产菌株，并进一步研究了前体供应对麦角硫因产量的影响，为以游动放线菌为底盘生产麦角硫因提供了策略支持。

麦角硫因  /  游动放线菌  /  高产菌株

[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.

ergothioneine  /  Actinoplanes sp.  /  high-yield strains
刘震, 常莹莹, 邓子新, 刘天罡. 麦角硫因在游动放线菌底盘中的高效合成. 微生物学报, 2024 , 64 (8) : 2752 -2767 . DOI: 10.13343/j.cnki.wsxb.20240025
Zhen LIU, Yingying CHANG, Zixin DENG, Tiangang LIU. Efficient synthesis of ergothioneine in Actinoplanes sp.[J]. Acta Microbiologica Sinica, 2024 , 64 (8) : 2752 -2767 . DOI: 10.13343/j.cnki.wsxb.20240025
正文
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麦角硫因(ergothioneine, EGT)，即2-巯基组氨酸三甲基甜菜碱，是一种由组氨酸衍生而来的含硫非蛋白源氨基酸[1]，1909年首次由Charles Tanret在研究麦角菌(Clavieps purpurea)时发现[2]。在包括人类在内的哺乳动物体内，其优先分布在肝脏、肾脏、红细胞、眼晶状体等易受高水平氧化应激和炎症影响的器官、细胞和分泌物中[3]。麦角硫因具有抗氧化、抗炎、抵抗紫外线辐射、护色等生理功能，同时也是一种重要的细胞生理保护剂[4]，被推荐为十大延年益寿的维生素之一[5]，其在食品、医药以及化妆品等领域有着广泛的应用。
人体不能够直接合成麦角硫因，只能通过特定的选择性转运蛋白从饮食中摄取并输送到各组织中[6]。目前麦角硫因主要通过3种方式获取：化学合成、食用菌提取和微生物发酵。其中化学合成法不仅工艺复杂，而且常常导致局部或全部的外消旋化，这无疑增加生产成本，而且容易产生带毒性的杂质，从而无法保障产品安全性[7]；而利用食用菌提取也存在着麦角硫因含量低、提取过程困难、生产周期长、成本高等问题[8]。工程菌株的构建和微生物发酵生产的方法，因其具有高产量且成本低的优点，已成为当前的研究热点。自然界中，麦角硫因可由蕈菌[9]、蓝藻细菌[10]、裂殖酵母[11]、放线菌[12]和甲基杆菌[1]等天然合成。多种微生物的麦角硫因生物合成途径已被鉴定，主要可分为以耻垢分枝杆菌(Mycobacterium smegmatis)为代表的细菌途径和以粗糙脉孢菌(Neurospora crassa)为代表的真菌途径[13-14]。如图1所示，在真菌中，麦角硫因的生物合成仅需要Egt1和Egt2两个酶，其中Egt1作为一种双功能酶，可以催化S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)上的甲基转移至l-组氨酸，合成l-组氨酸甜菜碱(l-hercynine, HER)[15]，
随后继续催化HER与半胱氨酸间C−S键的生成，形成组氨酸甜菜碱半胱氨酸亚砜(hercynine-cysteine-sulphoxide, HER-Cys-Sul)[16]，Egt2裂解HER-Cys-Sul中的C−S键，得到终产物麦角硫因[11]。在M. smegmatis中，麦角硫因的生成需要五步酶催化反应，这些酶由基因簇egtABCDE编码：EgtD以S-腺苷甲硫氨酸作为甲基供体将l-组氨酸甲基化形成HER；EgtA将谷氨酸与半胱氨酸连接合成γ-谷氨酰半胱氨酸(γ-glutamyl-cysteine, γ-Glu-Cys)；EgtB是一种单核非血红素铁酶，以γ-Glu-Cys为硫供体，可将HER转化为海西烯基-γ-谷氨酰半胱氨酸亚砜(hercynine-γ-glutamyl-cysteine-sulphoxide, HER-γ-Glu-Cys-Sul)；之后，谷氨酰胺酰胺水解酶EgtC除去HER-γ-Glu-Cys-Sul中的l-谷氨酸部分，生成HER-Cys-Sul；最后HER-Cys-Sul在磷酸吡哆醛依赖的碳硫裂解酶EgtE的作用下，裂解C−S键，形成终产物麦角硫因[17]。
根据其合成途径，研究者们通过代谢工程手段，实现了麦角硫因在不同底盘菌株中的合成与高产。Pluskal等[11]通过高表达Egt1，使粟酒裂殖酵母(Schizosaccharomyces pombe)中麦角硫因产量提升至368 mg/L。van der Hoek等[18]对酿酒酵母(Saccharomyces cerevisiae)的氨基酸代谢途径进行了设计，并在该工程菌株中组合表达了麦角硫因合成途径，采用不添加氨基酸的补料分批发酵培养，160 h麦角硫因的发酵产量可达2.4 g/L。Xiong等[19]在新金色分枝杆菌(Mycobacterium neoaurum)中通过增加前体l-组氨酸和甲基供体S-腺苷甲硫氨酸的供应，以及对麦角硫因降解酶进行失活，使得麦角硫因的产量提高到1.56 g/L。Hirasawa等[20]将耻垢分枝杆菌(Mycolicibacterium smegmatis)的EgtDE和甲基杆菌属(Methylobacterium sp.)的EgtB在谷氨酸棒状杆菌(Corynebacterium glutamicum)中进行表达，发酵2周后，其生产水平达到100 mg/L以上。另外，在解脂耶氏酵母(Yarrowia lipolytica)中表达来源于粗糙脉孢菌(Neurospora crassa)的生物合成酶Egt1和麦角菌(Claviceps purpurea)的Egt2后，在1 L生物反应器中进行磷酸盐限制补料分批发酵，220 h内产量达到1.63 g/L[21]。Zhang等[22]将一个组合的麦角硫因合成途径引入到大肠杆菌(Escherichia coli)中，并对参与麦角硫因合成的关键酶进行修饰，在5 L生物反应器中进行补料分批发酵，在添加氨基酸和甘油培养94 h后，麦角硫因产量达到5.4 g/L。
虽然在这些底盘菌株中通过引入麦角硫因合成过程中的相关基因，实现了麦角硫因的异源合成，但相比于异源表达，以能天然合成麦角硫因的菌株为底盘构建高产菌株，更有望突破底物耐受性的限制，在产量上有更高的提升空间。基于此，本研究以一种能够内源合成麦角硫因且生物安全性较高的游动放线菌(Actinoplanes sp.) HS为底盘，在E. coli BL21中异源表达，鉴定了其中的麦角硫因生物合成酶，并增加其对应合成基因的拷贝数，得到突变株YC313和YC314，其麦角硫因产量相较于出发菌株有了明显提升，此外也探究了在发酵过程中不同前体的添加对产量的影响。这为以放线菌为底盘生产麦角硫因的工业化应用提供了支持，也为通过增加前体供应进一步提高麦角硫因产量的改造策略提供了指导。
1 材料与方法
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1.1 材料
1.1.1 菌株和质粒
本研究所用的菌株、质粒和引物分别如表1和表2所示。Actinoplanes sp. HS为本实验室来源的一株能够内源合成麦角硫因的游动放线菌，其保藏编号为CCTCC No: M 2022390。
1.1.2 培养基
LB培养基(g/L)：胰蛋白胨10.0，酵母提取物5.0，NaCl 10.0，用于E. coli培养。
LA培养基(g/L)：胰蛋白胨10.0，酵母提取物5.0，NaCl 10.0，琼脂粉20.0，用于E. coli平板培养。
SFM固体培养基(g/L)：黄豆饼粉20.0，甘露醇20.0，琼脂16.0，pH 7.2，用于Actinoplanes sp. HS平板培养。
种子培养基(g/L)：葡萄糖10.0，黄豆饼粉40.0，甘油10.0，可溶性淀粉10.0，CaCO3 2.0，pH 7.0，用于Actinoplanes sp. HS及其突变株种子培养。
发酵培养基(g/L)：麦芽糖30.0，葡萄糖30.0，黄豆饼粉10.0，酵母粉3.0，K2HPO4·3H2O 1.0，FeCl3 1.5，CaCl2 2.5，CaCO3 2.5，pH 6.5，用于Actinoplanes sp. HS及其突变株发酵。
大肠杆菌发酵培养基(g/L)：葡萄糖20.0，酵母提取物2.0，(NH4)2SO4 16.0，Na2HPO4·12H2O 16.0，KH2PO4 3.0，MgSO4 0.6，CaCl2 0.01，pH 6.8−7.0，用于E. coli发酵。
1.2 基因组测序及分析
Actinoplanes sp. HS全基因组测序由武汉未来组生物科技有限公司使用PromethION测序平台完成，测序了1个cell，质控后的数据用flye (参数：--nano-raw)进行组装后，用racon (参数：default)结合三代测序数据进行矫正，用pilon (参数：default)和nextpolish (参数：default)结合二代测序数据进行校正。编码基因用prodigal (参数：−p None-g 11)进行预测，保留完整的编码序列(coding sequence, CDS)。利用直系同源基因簇(clusters of orthologous genes, COG)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)、基因本体论(gene onotology, GO)、美国国家生物技术信息中心参考序列数据库(NCBI reference sequence database)、蛋白质家族数据库(protein families database)和基因组研究所蛋白质家族数据库(the institute for genomic research’s database of protein families, TIGRFAMs)对编码蛋白进行功能注释。使用antiSMASH 3.0软件预测次级代谢产物合成基因簇。
1.3 质粒构建
pCIL001A质粒构建：以pETDuet-1质粒作为载体，使用Nco I/EcoR I双酶切得到片段化载体，以Actinoplanes sp. HS菌株基因组为模板，以Primer 1/Primer 2为引物扩增得到片段BC03-04016，以Primer 3/Primer 4为引物扩增得到片段BC03-04015，上述片段化载体和2个扩增片段使用多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司)进行组装，通过菌落PCR验证以及酶切验证后，将构建好的质粒进行测序，测序结果正确即pCIL001A质粒构建成功。
pCIL001质粒构建：以pCIL001A质粒作为载体，使用Nde I/Xho I双酶切得到片段化载体，以Actinoplanes sp. HS菌株基因组为模板，以Primer 5/Primer 6为引物扩增得到片段BC03-04014，以Primer 7/Primer 8为引物扩增得到片段BC03-04013，上述片段化载体和2个扩增片段使用多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司)进行组装，通过菌落PCR验证以及酶切验证后，将构建好的质粒进行测序，测序结果正确即pCIL001质粒构建成功。
pCIL003−pCIL008质粒构建：以pBBR1MCS-2质粒作为载体，使用Primer 9/Primer 10为引物扩增得到载体片段后，使用Nde I/Sac I双酶切，得到片段化的载体片段。以Actinoplanes sp. HS菌株基因组为模板，分别以Primer 11/Primer 12、Primer 26/Primer 27、Primer 28/Primer 29、Primer 30/Primer 31、Primer 32/Primer 33、Primer 13/Primer 14为引物扩增得到片段BC03-04046、BC03-00348、BC03-00103、BC03-05049、BC03-00781、BC03-04917，上述片段化载体分别与6个扩增片段使用多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司)进行组装，通过菌落PCR验证以及酶切验证后，将构建好的质粒进行测序，测序结果正确即得到质粒pCIL003、pCIL004、pCIL005、pCIL006、pCIL007、pCIL008。
pCIL013质粒构建：以pSET152质粒作为载体，使用Xba I/Eco32 I双酶切得到片段化的载体片段，标记为152-XE，以pIB139质粒为模板，Primer 15/Primer 16为引物扩增得到启动子片段ermE*(1)，以Actinoplanes sp. HS菌株基因组为模板，Primer 17/Primer 18为引物扩增得到片段BC03-04016-BC03-04015-BC03-04014-BC03-04013；以pIB139质粒为模板，Primer 19/Primer 20为引物扩增得到启动子片段ermE*(2)，以Actinoplanes sp. HS菌株基因组为模板，Primer 21/Primer 22为引物扩增得到片段BC03-04046，上述扩增片段ermE*(1)和BC03-04016-BC03-04015-BC03-04014-BC03-04013使用Primer 15/Primer 18引物进行重叠延伸PCR (overlap extension polymerase chain reaction, OE-PCR)，得到拼接片段ermE*-BC03-04016-BC03-04015-BC03-04014-BC03-04013，标记为K16-13，扩增片段ermE*(2)和BC03-04046使用Primer 19/Primer 22引物进行OE-PCR得到拼接片段ermE*-BC03-04046，标记为E-46。上述片段152-XE、K16-13和E-46使用多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司)进行组装，通过菌落PCR验证以及酶切验证后，将构建好的质粒进行测序，测序结果正确即pCIL013质粒构建成功。
pCIL014质粒构建：以pIB139质粒为模板，Primer 19/Primer 23为引物扩增得到启动子片段ermE*(3)，以Actinoplanes sp. HS菌株基因组为模板、Primer 24/Primer 25为引物扩增得到片段BC03-04917，上述扩增片段ermE*(3)和BC03-04917使用Primer 19/Primer 25引物进行OE-PCR得到拼接片段ermE*-BC03-04917，标记为E-17。上述片段152-XE、K16-13和E-17使用多片段一步法快速克隆试剂盒(上海翊圣生物科技有限公司)进行组装，通过菌落PCR验证以及酶切验证后，将构建好的质粒进行测序，测序结果正确即pCIL014质粒构建成功。
1.4 突变株构建
1.4.1 E. coli BL21(DE3)中异源表达菌株的获得
取适量E. coli BL21(DE3)甘油菌于LA平板分离单菌落，挑取单菌落于10 mL LB培养基中，37 ℃、220 r/min振荡培养过夜，以1%转接量转接至200 mL LB培养基中，37 ℃、220 r/min振荡培养至OD600为0.4−0.6，收集菌液，冰上预冷10 min，4 ℃、3 500 r/min离心10 min，去上清，加30 mL CCC1溶液(30 mmol/L KAc，80 mmol/L CaCl2，pH值约5.8)重悬，4 ℃、3 500 r/min离心10 min，去上清，将细胞重悬于30 mL CCC1中，冰浴1−2 h，4 ℃、3 500 r/min离心10 min，去上清，将细胞重悬于6 mL CCC2 (CCC1+10%甘油)中，每个1.5 mL无菌离心管中分装70 μL，液氮速冻，即得E. coli BL21(DE3)感受态细胞。
将pCIL001和pETDuet-1分别通过钙转法转化入E. coli BL21(DE3)感受态中，得到菌株YC301和YC300，其中，YC301为异源表达菌株，YC300为对照菌株。
取上述得到的YC301菌株的甘油菌于含有100 μg/mL氨苄青霉素的LA平板分离单菌落，挑取单菌落于10 mL含有100 μg/mL氨苄青霉素的LB培养基中，37 ℃、220 r/min振荡培养过夜。以1%转接量转接至200 mL含有100 μg/mL氨苄青霉素的LB培养基中，37 ℃、220 r/min振荡培养至OD600为0.4−0.6，收集菌液，后续YC301菌株感受态细胞制备过程同E. coli BL21(DE3)感受态细胞制备过程。
将构建的pCIL003、pCIL008质粒分别通过钙转法转化入至YC301菌株感受态中，得到菌株YC303和YC308。
1.4.2 Actinoplanes sp. HS中高表达菌株的获得
pCIL013、pCIL014质粒测序验证正确后，分别转化至ET12567-pUZ8002菌株中，挑取单克隆于含有50 μg/mL安普霉素、50 μg/mL卡那霉素、25 μg/mL氯霉素的LB液体培养基中，37 ℃、220 r/min培养过夜，过夜培养菌液以1/100 (体积比)转接至4 mL含50 μg/mL安普霉素、50 μg/mL卡那霉素、25 μg/mL氯霉素的LB液体培养基中，37 ℃、220 r/min培养至OD600为0.6−0.8，离心收集菌体，等体积无抗LB液体培养基洗涤菌体3次后，用100 μL无抗LB液体培养基重悬，得大肠杆菌悬浮液，作为供体菌。收集SFM固体培养基28 ℃培养7 d的Actinoplanes sp. HS菌体，30 mL LB液体培养基洗涤菌体2次，3 mL无抗LB液体培养基重悬，作为受体菌。
将上述100 μL供体菌和100 μL受体菌悬液混合，均匀涂布于含有10 mmol/L MgCl2的SFM固体培养基上，28 ℃培养16−18 h，使用安普霉素(25 μg/mL)和甲氧苄啶(40 μg/mL)覆盖平板，28 ℃继续培养7 d，长出的接合子于含有50 μg/mL安普霉素的SFM固体培养基上分离单菌落，单菌落涂布于含有50 μg/mL安普霉素的SFM平板上，长出的菌落取适量于50 μL ddH2O中，沸水煮5 min后冰浴5 min，重复3次，短暂离心后取上清液进行PCR验证，验证正确突变株命名为YC313和YC314。
1.5 菌株发酵
1.5.1 E. coli BL21(DE3)中异源表达菌株发酵
取YC300、YC301菌株分别于含100 μg/mL氨苄青霉素的LA平板分离单菌落，分别挑取其单克隆于10 mL含有100 μg/mL氨苄青霉素的LB液体培养基中，37 ℃、220 r/min培养过夜，以1%转接量转接至50 mL含有100 μg/mL氨苄青霉素的大肠杆菌发酵培养基中，37 ℃、220 r/min培养至OD600值约为0.8，加入终浓度0.2 mmol/L的IPTG诱导，改变温度至25 ℃、220 r/min继续培养120 h。
YC303、YC308菌株分别于含有100 μg/mL氨苄青霉素和50 μg/mL卡那霉素的LA平板分离单菌落，分别挑取其单克隆于10 mL含有100 μg/mL氨苄青霉素和50 μg/mL卡那霉素的LB液体培养基中，37 ℃、220 r/min培养过夜，以1% 转接量转接至含有100 μg/mL氨苄青霉素和50 μg/mL卡那霉素的发酵培养基(同YC301菌株的发酵培养基)中，37 ℃、220 r/min培养至OD600值约为0.8，加入终浓度0.2 mmol/L IPTG诱导，改变温度至25 ℃、220 r/min继续培养120 h，在诱导后24、48和120 h取样。
1.5.2 Actinoplanes sp. HS及突变株发酵
取Actinoplanes sp. HS的甘油菌于SFM固体培养基分离单菌落，28 ℃培养5−7 d，单菌落长出后，挑取单菌落于SFM固体培养基上涂板培养，28 ℃培养7 d。从生长7 d的SFM固体培养基上刮取菌体(1.5 cm×1.5 cm)接入25 mL种子培养基中，28 ℃、250 r/min振荡培养48 h，将生长48 h的种子液，取2.5 mL接入25 mL发酵培养基中，28 ℃、250 r/min振荡培养7 d，获得发酵液。
1.6 产量检测
1.6.1 发酵液中产物鉴定
大肠杆菌发酵液产物检测：取菌株120 h发酵液1 mL，10 000 r/min离心5 min，所得上清液使用0.22 μm滤膜过滤后直接进行高效液相色谱-高分辨质谱(high performance liquid chromatography-high resolution mass spectrometry, HPLC-HRMS)检测，菌体使用1 mL 50%乙腈重悬，600 W超声破碎30 min后，10 000 r/min离心5 min，所得上清液使用0.22 μm滤膜过滤后进行HPLC-HRMS检测。
放线菌发酵液产物检测：取发酵7 d后的发酵液5 mL，加入5 mL纯乙腈，混合均匀，600 W超声破碎30 min，4 000 r/min离心15 min，所得上清液稀释10倍后使用0.22 μm滤膜过滤并使用HPLC-HRMS进行检测。
HPLC-HRMS检测方法：色谱柱为Thermo Scientific Hypersil GOLD aQ (150 mm×2.1 mm, 3 μm)；流动相：A相为0.1%甲酸的水溶液，B相为0.1%甲酸的乙腈溶液，洗脱梯度：0−1 min，A相: B相=99:1；1−10 min，A相99%−10%，B相1%−90%；10−15 min，A相: B相=10:90；15−15.5 min，A相10%−99%，B相90%−1%；15.5−20 min，A相: B相=99:1；流速0.2 mL/min；进样体积2 μL。
1.6.2 产物定量分析
发酵液中麦角硫因定量使用高效液相色谱-紫外可见分光度检测器检测，检测方法如下：色谱柱为Dionex AcclaimTM 120 C18 (250 mm× 4.6 mm, 5 μm)；流动相：A相为纯乙腈溶液，B相为纯水溶液，洗脱梯度：0−15 min，A相: B相=3:97；15−18 min，A相3%−100%，B相97%−0%；18−19 min，A相100%，B相0%；19−20 min，A相100%−0%，B相0%−97%；20−30 min，A相: B相=3:97；流速0.8 mL/min；进样体积20 μL。用麦角硫因标准品(上海阿拉丁生化科技股份有限公司)绘制标准曲线进行定量。
2 结果与分析
收起
2.1 Actinoplanes sp. HS中麦角硫因合成酶的鉴定
2.1.1 基因筛选
研究指出egtABCDE组成的麦角硫因合成基因簇仅存在于放线菌门，而且大部分属于弗兰克氏菌亚目和棒杆菌亚目的菌株含有EgtE同源蛋白，属于小单孢菌亚目、丙酸杆菌亚目、假诺卡氏菌亚目、链霉菌亚目和链孢子囊亚目的菌株缺失egtE基因[23]。以M. smegmatis MC2 155的麦角硫因合成基因egtABCDE为比对序列，使用交互的BLASTp[24]来搜索Actinoplanes sp. HS基因组中分别对应的可能同源蛋白，通过生物信息学预测分析，寻找到可能参与麦角硫因合成的酶的编码基因，如表3所示。Actinoplanes sp. HS中具有egtA、egtB、egtC、egtD功能基因，这4个基因在Actinoplanes sp. HS基因组上成簇排列，其中BC03-04015和BC03-04013分别与egtB和etgD具有较高的同源性。然而通过比对，未发现与egtE功能完全一致的基因。
2.1.2 基因功能鉴定
为了进一步确定Actinoplanes sp. HS基因组中比对出的可能参与麦角硫因合成的基因的功能，将其在异源宿主E. coli BL21(DE3)中进行了表达，随后进行了摇瓶发酵和产物测定。
首先对麦角硫因前体HER-Cys-Sul合成相关酶的功能进行了鉴定，将基因BC03-04016、BC03-04015、BC03-04014、BC03-04013克隆至载体pETDuet-1上得到质粒pCIL001，进而将质粒转入E. coli BL21(DE3)中得到异源表达菌株YC301，同时以空载体转化得到的菌株YC300作为对照。对这2个菌株进行发酵后用高分辨质谱检测产物，如图2所示。结果表明，对照菌株YC300不能合成中间体HER-Cys-Sul，而异源表达BC03-04016、BC03-04015、BC03-04014、BC03-04013的菌株YC301可产生HER-Cys-Sul，从而证实Actinoplanes sp. HS中基因BC03-04016、BC03-04015、BC03-04014、BC03-04013所编码的蛋白(酶)可以合成麦角硫因前体HER-Cys-Sul。
麦角硫因合成的最后一步为HER-Cys-Sul在磷酸吡哆醛依赖的碳硫裂解酶EgtE的作用下，裂解C−S键[20]，并获得终产物麦角硫因。将比对到的5个候选基因BC03-04046、BC03-00348、BC03-00103、BC03-05049、BC03-00781、BC03-04917分别进行碳硫裂解酶的功能鉴定。将其分别克隆至载体pBBR1MCS-2上得到质粒pCIL003、pCIL004、pCIL005、pCIL006、pCIL007、pCIL008，将质粒分别转入YC301中得到菌株YC303、YC304、YC305、YC306、YC307、YC308，以YC301作为对照进行摇瓶发酵和产物测定。
对发酵液上清和菌体中检测发现，在YC301菌株中表达BC03-04046 (YC303)、BC03-04917 (YC308)后，中间体HER-Cys-Sul已检测不到，而在对照菌株YC301以及表达BC03-00348 (YC304)、BC03-00103 (YC305)、BC03-05049 (YC306)、BC03-00781 (YC307)后仍可以检测到中间体HER-Cys-Sul。通过二级质谱碎片比对，如图3C所示，表明YC303菌株和YC308菌株中生成了麦角硫因。YC303菌株和YC308菌株中不同时间发酵液的上清液和菌体中麦角硫因含量如图3D所示，YC303、YC308菌株发酵液中有麦角硫因积累。该结果表明基因BC03-04046和BC03-04917编码的酶具有碳硫裂解酶功能，可以催化前体HER-Cys-Sul形成麦角硫因。
2.2 麦角硫因高产菌株的获得
通过增加合成过程中关键基因的拷贝数是提高天然化合物产量常用的改造策略之一，因此为有效提升麦角硫因产量，进行了Actinoplanes sp. HS中麦角硫因合成相关基因的高表达。将已鉴定的麦角硫因前体HER-Cys-Sul合成相关基因BC03-04016-BC03-04015-BC03-04014-BC03-04013与具有碳硫裂解酶功能的基因BC03-04046和BC03-04917分别组合后构建至载体pSET152上，得到质粒pCIL013和pCIL014，然后使用两亲本接合转移通过attB/attP位点整合到Actinoplanes sp. HS中，得到高表达菌株YC313和YC314。将其进行发酵并以Actinoplanes sp. HS作为对照，结果如图4所示，YC313、YC314菌株麦角硫因产量分别达到125 mg/L和108 mg/L，较出发菌株Actinoplanes sp. HS的麦角硫因产量(43 mg/L)分别提高至2.9倍和2.5倍。
2.3 前体及底物添加
组氨酸、半胱氨酸和甲硫氨酸是直接参与麦角硫因合成的重要氨基酸。此外，O-乙酰高丝氨酸(O-acetylhomoserine)是麦角硫因底物SAM和半胱氨酸的合成前体，在O-乙酰高丝氨酸合成过程中，乙酰辅酶A (acetyl coenzyme A, acetyl-CoA)起着关键作用，而泛酸又是合成辅酶A (coenzyme A, CoA)过程中所必需的[25]。基于此，尝试了在Actinoplanes sp. HS发酵培养基中添加不同浓度的麦角硫因合成前体组氨酸、半胱氨酸、甲硫氨酸以及泛酸钙，以探究Actinoplanes sp. HS中前体供应对麦角硫因产量的影响。以2.2获得的高产菌株YC313作为发酵菌株，以1.1.2中发酵培养基作为对照，在发酵2 d和7 d时分别测定发酵液产量，结果如图5A所示，在添加了半胱氨酸和甲硫氨酸后，7 d产量均有了一定程度的提升，在甲硫氨酸添加量为0.35 g/L时，相较于原始培养基产量提升幅度最大，提高了24%，而在添加0.01 g/L的泛酸钙后，产量有近7%的提升，但是在添加组氨酸后，产量并没有提高，反而有一定程度的降低。这些结果表明，在该菌株麦角硫因合成过程中的组氨酸供给可能是充足的，而半胱氨酸和甲硫氨酸的供应可能不够。
此外，在发酵过程中，发酵培养基中的黄豆饼粉为菌株氨基酸，蛋白质、核酸等含氮物质代谢提供了主要的氮源，本研究对其含量进行了优化。如图5B所示，在原始培养基中额外添加不同含量的黄豆饼粉进行比较，结果表明，当黄豆饼粉添加量达到10 g/L时，相比于原始培养基产量有了19%的提升。这表明原始培养基中的氮源可能不足。
3 讨论与结论
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麦角硫因作为一种稀有的天然氨基酸类强抗氧化剂[1]，在机体内发挥着重要的生理功能[4]，在食品、医药、化妆品领域有着广泛的应用。传统的从蕈菇中提取和化学合成的方法存在收率低、成本高等问题[8]，利用工程菌株的构建和微生物发酵生产的方法可以很好地解决这一问题。
基于此，本研究以一种可以天然生产麦角硫因的菌株Actinoplanes sp. HS为出发菌株，通过生物信息学分析以及在大肠杆菌E. coli BL21(DE3)中异源表达，对中间体和终产物进行检测，鉴定了该菌株中麦角硫因合成的相关基因BC03-04016、BC03-04015、BC03-04014、BC03-04013、BC03-04046和BC03-04917的功能。其中基因BC03-04016、BC03-04015、BC03-04014、BC03-04013所编码的酶可以合成麦角硫因前体HER-Cys-Sul，基因BC03-04046和BC03-04917都具有碳硫裂解酶功能，可以催化前体HER-Cys-Sul形成终产物麦角硫因。随后，将BC03-04016-BC03-04015-BC03-04014-BC03-04013和2种碳硫裂解酶基因BC03-04046、BC03-04917分别组合后，在Actinoplanes sp. HS中进行高表达，得到突变株YC313和YC314，其对应的麦角硫因产量为125 mg/L和108 mg/L，较出发菌株Actinoplanes sp. HS的产量(43 mg/L)分别提高至2.9倍和2.5倍。
通过培养基中不同浓度的麦角硫因合成前体添加实验发现，在添加了不同含量的泛酸钙、半胱氨酸和甲硫氨酸后，产量均有一定程度的提升，在甲硫氨酸添加量为0.35 g/L时，麦角硫因产量相较于原始培养基产量提升幅度最大，进一步提高了24%，这表明在该菌株的麦角硫因合成过程中，前体物质半胱氨酸和甲硫氨酸的供应可能不足。因此，后续可考虑对氨基酸代谢途径进行理性设计，增加半胱氨酸和甲硫氨酸的供应，有望实现产量的进一步提升。此外，在原始发酵培养基中额外添加10 g/L的氮源黄豆饼粉后发现，麦角硫因的产量也提升了19%，这表明继续对发酵培养基进行优化，麦角硫因的产量仍有进一步提升的空间。综上所述，本研究为以游动放线菌为底盘生产麦角硫因提供了依据，也为在其他内源生产麦角硫因的菌株中进行代谢工程改造的策略提供了借鉴。
基金
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  • 武汉合生科技有限公司创新研发基金(T20220101)
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doi: 10.13343/j.cnki.wsxb.20240025
  • 接收时间：2024-01-08
  • 首发时间：2026-03-19
  • 出版时间：2024-03-12
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  • 收稿日期：2024-01-08
  • 录用日期：2024-03-07
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Wuhan Hesheng Technology Co., Ltd. Innovation Fund(T20220101)
武汉合生科技有限公司创新研发基金(T20220101)
作者信息
    1 武汉大学药学院 组合生物合成与新药发现教育部重点实验室, 湖北 武汉 430071
    2 上海交通大学生命科学技术学院 微生物代谢国家重点实验室, 上海 200240

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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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