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Article(id=1242175011402903708, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1242175008705966230, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240504, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1723651200000, receivedDateStr=2024-08-15, revisedDate=null, revisedDateStr=null, acceptedDate=1730217600000, acceptedDateStr=2024-10-30, onlineDate=1774087201213, onlineDateStr=2026-03-21, pubDate=1735920000000, pubDateStr=2025-01-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1774087201213, onlineIssueDateStr=2026-03-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1774087201213, creator=13701087609, updateTime=1774087201213, updator=13701087609, issue=Issue{id=1242175008705966230, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='1', pageStart='1', pageEnd='415', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1774087200568, creator=13701087609, updateTime=1774087310368, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1242175469299270453, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1242175008705966230, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1242175469299270454, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1242175008705966230, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=256, endPage=267, ext={EN=ArticleExt(id=1242175018935873779, articleId=1242175011402903708, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Interaction between type Ⅰ signal peptidase and signal peptides in Bacillus amyloliquefaciens, columnId=1241045257748533520, journalTitle=Acta Microbiologica Sinica, columnName=Research Articles, runingTitle=null, highlight=null, articleAbstract=

[Objective] Based on the critical role of type Ⅰ signal peptidase in the secretion system, this study explores the interaction between signal peptidase and signal peptides to guide the optimization of aminopeptidase secretion expression in Bacillus amyloliquefaciens. [Methods] The endogenous signal peptidase and signal peptide of Bacillus amyloliquefaciens TCCC 19030 were examined using relative fluorescence intensity and enzyme activity for analysis, and molecular docking to study their interaction. [Results] The signal peptide YolC fused with aminopeptidase exhibited the highest extracellular enzyme activity, reaching 11 847.67 U/mL. Overexpression of the signal peptidase SipW increased aminopeptidase activity to 16 261 U/mL. Molecular docking results also showed that YolC had the lowest binding free energy with SipW, at −4.4 kcal/mol. [Conclusion] Optimization of signal peptides and overexpression of signal peptidase can effectively enhance the secretion of aminopeptidase. The binding energy between signal peptidase and signal peptide is a key factor influencing the secretion levels of the target protein.

, correspAuthors=Yu LI, authorNote=null, correspAuthorsNote=
*LI Yu E-mail:
, copyrightStatement=Copyright ©2025 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=Jiejie GUO, Weihong WANG, Jiemin HE, Dengke LI, Yexue LIU, Fuping LU, Yu LI), CN=ArticleExt(id=1242175024883397275, articleId=1242175011402903708, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=解淀粉芽孢杆菌Ⅰ型信号肽酶与信号肽的相互作用, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

【目的】基于Ⅰ型信号肽酶在分泌系统中的重要作用，探究信号肽酶与信号肽之间的作用关系，指导氨肽酶在解淀粉芽孢杆菌中的分泌表达优化。【方法】对解淀粉芽孢杆菌TCCC 19030的内源性信号肽酶和信号肽进行筛选，采用相对荧光强度、酶活力等表征手段进行分析，同时采用分子对接技术探究信号肽酶和信号肽的相互作用关系。【结果】信号肽YolC融合氨肽酶的胞外酶活力最高，达到11 847.67 U/mL。过表达信号肽酶SipW后，氨肽酶的酶活力提高到16 261 U/mL。分子对接结果显示，信号肽YolC与信号肽酶SipW结合自由能最低，为−4.4 kcal/mol。【结论】信号肽优化与信号肽酶过表达可有效提高氨肽酶的分泌表达，其中信号肽酶与信号肽的结合能是影响目的蛋白分泌量的关键因素之一。

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Tianjin: Master's Thesis of Tianjin University of Science & Technology, 2022 (in Chinese)., articleTitle=null, refAbstract=null), Reference(id=1243300013049233550, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[23], rfOrder=29, authorNames=null, journalName=null, refType=null, unstructuredReference=吴怡. 以细菌群体感应系统为靶向的噁唑烷酮类抗毒力药物筛选与功效研究[D]. 成都: 成都大学硕士学位论文, 2024., articleTitle=null, refAbstract=null), Reference(id=1243300013179256981, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[23], rfOrder=30, authorNames=null, journalName=null, refType=null, unstructuredReference=WU Y. Screening and efficacy study of oxazolidinone anti-virulence drugs targeting bacterial quorum sensing systems[D]. Chengdu: Master's Thesis of Chengdu University, 2024 (in Chinese)., articleTitle=null, refAbstract=null)], funds=[Fund(id=1243300006178964384, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, awardId=2021YFC2104000, language=EN, fundingSource=National Key Research and Development Program of China(2021YFC2104000), fundOrder=null, country=null), Fund(id=1243300006271239078, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, awardId=2021YFC2104000, language=CN, fundingSource=国家重点研发计划(2021YFC2104000), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1243299996649505090, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, xref=null, ext=[AuthorCompanyExt(id=1243299996670476613, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, companyId=1243299996649505090, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, School of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China), AuthorCompanyExt(id=1243299996674670919, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, companyId=1243299996649505090, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=天津科技大学 生物工程学院, 工业发酵微生物教育部重点实验室, 天津 300457)])], figs=[ArticleFig(id=1243300001602978467, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 1, caption=SDS-PAGE analysis of the extracellular proteins. 15% SDS-PAGE analysis of the fermentation supernatant after concentrated by ultrafiltration. Lane M: Protein Molecular Weight Marker; Lane 19030-YwaD: Bacillus amyloliquefaciens harboring pLY-3-YwaD plasmid; Lane 19030-NC: Bacillus amyloliquefaciens harboring pLY-3 plasmid., figureFileSmall=tpch6qaf26OyE3nWl8eV3A==, figureFileBig=yJljpmTq9TtR6SYKaMzLIw==, tableContent=null), ArticleFig(id=1243300001712030383, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图1, caption=胞外蛋白SDS-PAGE分析, figureFileSmall=tpch6qaf26OyE3nWl8eV3A==, figureFileBig=yJljpmTq9TtR6SYKaMzLIw==, tableContent=null), ArticleFig(id=1243300001833665211, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 2, caption=Enzyme activity of aminopeptidase guided by different signal peptides., figureFileSmall=XV4hupvePzFpl36zP9h/Nw==, figureFileBig=F9KgS2kG8BOvXURwzP4CrA==, tableContent=null), ArticleFig(id=1243300003347808961, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图2, caption=不同信号肽融合氨肽酶的酶活力, figureFileSmall=XV4hupvePzFpl36zP9h/Nw==, figureFileBig=F9KgS2kG8BOvXURwzP4CrA==, tableContent=null), ArticleFig(id=1243300003452666567, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 3, caption=Relative position of type Ⅰ signal peptidase on the Bacillus amyloliquefaciens TCCC 19030 genome., figureFileSmall=xiz5HbHBDbuuvEVjbLc73g==, figureFileBig=2/YgAUKbiaVr3NzOHM0EDA==, tableContent=null), ArticleFig(id=1243300003574301395, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图3, caption=Ⅰ型信号肽酶在Bacillus amyloliquefaciens TCCC 19030基因组上的相对位置, figureFileSmall=xiz5HbHBDbuuvEVjbLc73g==, figureFileBig=2/YgAUKbiaVr3NzOHM0EDA==, tableContent=null), ArticleFig(id=1243300003725296346, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 4, caption=Growth curves of type Ⅰ signal peptidase deletion strains., figureFileSmall=yBTV0nDIwFRK5TYm14v85w==, figureFileBig=p3KtCoOCCwq3Wnm1UvKk5A==, tableContent=null), ArticleFig(id=1243300003846931172, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图4, caption=Ⅰ型信号肽酶缺失菌株生长曲线, figureFileSmall=yBTV0nDIwFRK5TYm14v85w==, figureFileBig=p3KtCoOCCwq3Wnm1UvKk5A==, tableContent=null), ArticleFig(id=1243300003951788777, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 5, caption=Enzyme activity of aminopeptidase in signal peptidase-deficient strains., figureFileSmall=AcoAO8W5uibIjhtCdeqkbg==, figureFileBig=bboo9AyYPGwv9HQvvRlbcw==, tableContent=null), ArticleFig(id=1243300004098589431, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图5, caption=氨肽酶在信号肽酶缺失菌株中的酶活力, figureFileSmall=AcoAO8W5uibIjhtCdeqkbg==, figureFileBig=bboo9AyYPGwv9HQvvRlbcw==, tableContent=null), ArticleFig(id=1243300004249584385, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 6, caption=Aminopeptidase activity of signal peptidase genes overexpressed., figureFileSmall=MIYNJvP4u6dULlv6JpTufA==, figureFileBig=VzeIy0rvi0D0PJl+XPFknw==, tableContent=null), ArticleFig(id=1243300004375413518, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图6, caption=信号肽酶基因过表达的氨肽酶酶活力, figureFileSmall=MIYNJvP4u6dULlv6JpTufA==, figureFileBig=VzeIy0rvi0D0PJl+XPFknw==, tableContent=null), ArticleFig(id=1243300004589323035, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 7, caption=SDS-PAGE analysis of extracellular proteins from 19030-YwaD-SipA, 19030-YwaD-SipS, 19030-YwaD-SipW, 19030-YwaD-SipV, and BY-19030. Lane M: Protein Molecular Weight Marker; Lane 1: BY-19030; Lane 2: 19030-YwaD-SipA; Lane 3: 19030-YwaD-SipS; Lane 4: 19030-YwaD-SipV; Lane 5: 19030-YwaD-SipW., figureFileSmall=lIxJYMZTliM9pohYs5qyEw==, figureFileBig=xu4PBDgTTFN2hFkhcqcQmw==, tableContent=null), ArticleFig(id=1243300004740317990, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图7, caption=过表达信号肽酶基因对氨肽酶影响的SDS-PAGE分析, figureFileSmall=lIxJYMZTliM9pohYs5qyEw==, figureFileBig=xu4PBDgTTFN2hFkhcqcQmw==, tableContent=null), ArticleFig(id=1243300004849369902, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 8, caption=Model of signal peptide YolC and models of signal peptidases. A: Model of proline-asparagine-lysine; B: SipA prediction model; C: SipS prediction model; D: SipV prediction model; E: SipW prediction model., figureFileSmall=m39Q/4n6iKu9mn/53EMUdQ==, figureFileBig=EqXylGGlxkFAJp+qV++tiw==, tableContent=null), ArticleFig(id=1243300004962616119, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图8, caption=信号肽YolC末端的三肽模型与信号肽酶的结构模型。

A：脯氨酸-天冬酰胺-赖氨酸三肽的模型；B：SipA预测模型；C：SipS预测模型；D：SipV预测模型；E：SipW预测模型。

, figureFileSmall=m39Q/4n6iKu9mn/53EMUdQ==, figureFileBig=EqXylGGlxkFAJp+qV++tiw==, tableContent=null), ArticleFig(id=1243300005084250949, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 9, caption=The docking models of P-N-K and signal peptidases. A: The docking model of P-N-K and SipA; B: The docking model of P-N-K and SipS; C: The docking model of P-N-K and SipV; D: The docking model of P-N-K and SipW., figureFileSmall=OMS7gOERd+ce5kXKH387CQ==, figureFileBig=BgdjwAKBrkAqmi1jQQeU2g==, tableContent=null), ArticleFig(id=1243300005210080080, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图9, caption=P-N-K与信号肽酶的对接模型。

A：P-N-K和SipA的对接模型；B：P-N-K和SipS的对接模型；C：P-N-K和SipV的对接模型；D：P-N-K和SipW的对接模型。

, figureFileSmall=OMS7gOERd+ce5kXKH387CQ==, figureFileBig=BgdjwAKBrkAqmi1jQQeU2g==, tableContent=null), ArticleFig(id=1243300005306549079, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Figure 10, caption=The binding free energies of signal peptide YolC and different signal peptidases analog docking., figureFileSmall=6XetU1KuXVRo4MPDvKpqkw==, figureFileBig=N6G/VU9IDGxyzz4SCY1zwQ==, tableContent=null), ArticleFig(id=1243300005444961123, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=图10, caption=信号肽YolC与不同信号肽酶模拟对接的结合自由能, figureFileSmall=6XetU1KuXVRo4MPDvKpqkw==, figureFileBig=N6G/VU9IDGxyzz4SCY1zwQ==, tableContent=null), ArticleFig(id=1243300005545624429, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Table 1, caption=

The strains and plasmids used in the study

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsRelevant characteristicsSources
Strains
  B. amyloliquefaciens TCCC 19030B. amyloliquefaciens, used for initial expression hostThis lab
  E. coli JM109Host for plasmid constructionThis lab
  E. coli EC135/pM.BamHost for plasmid-methylated modificationInstitute of Microbiology, Chinese Academy of Sciences
  BY-19030B. amyloliquefaciens harboring pLY-3-YolC-YwaD plasmidThis study
  19030-YwaDB. amyloliquefaciens harboring pLY-3-YwaD plasmidThis study
  19030-NCB. amyloliquefaciens harboring pLY-3 plasmidThis study
  19030-ΔWB. amyloliquefaciens, ΔsipWThis study
  19030-ΔAB. amyloliquefaciens, ΔsipAThis study
  19030-ΔSB. amyloliquefaciens, ΔsipSThis study
  19030-ΔVB. amyloliquefaciens, ΔsipVThis study
  BY-ΔA19030-ΔA harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔV19030-ΔV harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔS19030-ΔS harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔW19030-ΔW harboring pLY-3-YolC-YwaD plasmidThis study
  19030-YwaD-SipWB. amyloliquefaciens, sipW gene overexpressionThis study
  19030-YwaD-SipAB. amyloliquefaciens, sipA gene overexpressionThis study
  19030-YwaD-SipSB. amyloliquefaciens, sipS gene overexpressionThis study
  19030-YwaD-SipVB. amyloliquefaciens, sipV gene overexpressionThis study
Plasmids
  pWH-T2Temperature-sensitive shuttle vector, KanarHubei University
  pLY-3Shuttle expression vector, Cmr (E. coli) and Kanar (Bacillus), MCSThis lab
  pLY-3-YolC-YwaDAminopeptidase expression vector, KanarThis study
  pWH-T2-SipWKnockdown plasmid, sipW gene deletion, KanarThis study
  pWH-T2-SipAKnockdown plasmid, sipA gene deletion, KanarThis study
  pWH-T2-SipSKnockdown plasmid, sipS gene deletion, KanarThis study
  pWH-T2-SipVKnockdown plasmid, sipV gene deletion, KanarThis study
  pWH-SipW-YwaDsipV gene and ywaD gene expression vector, KanarThis study
), ArticleFig(id=1243300005717590904, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=表1, caption=

本研究中所用的菌株和质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsRelevant characteristicsSources
Strains
  B. amyloliquefaciens TCCC 19030B. amyloliquefaciens, used for initial expression hostThis lab
  E. coli JM109Host for plasmid constructionThis lab
  E. coli EC135/pM.BamHost for plasmid-methylated modificationInstitute of Microbiology, Chinese Academy of Sciences
  BY-19030B. amyloliquefaciens harboring pLY-3-YolC-YwaD plasmidThis study
  19030-YwaDB. amyloliquefaciens harboring pLY-3-YwaD plasmidThis study
  19030-NCB. amyloliquefaciens harboring pLY-3 plasmidThis study
  19030-ΔWB. amyloliquefaciens, ΔsipWThis study
  19030-ΔAB. amyloliquefaciens, ΔsipAThis study
  19030-ΔSB. amyloliquefaciens, ΔsipSThis study
  19030-ΔVB. amyloliquefaciens, ΔsipVThis study
  BY-ΔA19030-ΔA harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔV19030-ΔV harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔS19030-ΔS harboring pLY-3-YolC-YwaD plasmidThis study
  BY-ΔW19030-ΔW harboring pLY-3-YolC-YwaD plasmidThis study
  19030-YwaD-SipWB. amyloliquefaciens, sipW gene overexpressionThis study
  19030-YwaD-SipAB. amyloliquefaciens, sipA gene overexpressionThis study
  19030-YwaD-SipSB. amyloliquefaciens, sipS gene overexpressionThis study
  19030-YwaD-SipVB. amyloliquefaciens, sipV gene overexpressionThis study
Plasmids
  pWH-T2Temperature-sensitive shuttle vector, KanarHubei University
  pLY-3Shuttle expression vector, Cmr (E. coli) and Kanar (Bacillus), MCSThis lab
  pLY-3-YolC-YwaDAminopeptidase expression vector, KanarThis study
  pWH-T2-SipWKnockdown plasmid, sipW gene deletion, KanarThis study
  pWH-T2-SipAKnockdown plasmid, sipA gene deletion, KanarThis study
  pWH-T2-SipSKnockdown plasmid, sipS gene deletion, KanarThis study
  pWH-T2-SipVKnockdown plasmid, sipV gene deletion, KanarThis study
  pWH-SipW-YwaDsipV gene and ywaD gene expression vector, KanarThis study
), ArticleFig(id=1243300005839225733, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=EN, label=Table 2, caption=

Primers used in the study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers nameSequences (5′→3′)
For the construction of expression cassette of SipW
  sipW-Up-FCCCTTAACGAATTCCTGCAGCCCCTGATACGTCAGGCACCGTG
  sipW-Up-RAATCAAGCTGAAGATTACGGCATAC
  sipW-Down-FGTATGCCGTAATCTTCAGCTTGATTCATGAGAGAAACGGGACCATGTC
  sipW-Down-RCACCGCGGTGGCGGCCGCTCTAGTCTTTACCGACTGTCAGAACCG
  sipW-djh-FCCGACTGCGCAAAAGACATAATC
  sipW-djh-RCTGCCTGAGGCAGCATTAAC
  sipW-sjh-FGTGGTGGCGAATACGAATGC
  sipW-sjh-RCTGCCTGAGGCAGCATTAAC
  sipW-FATGGGAGGAAATCACTTGAAATCAG
  sipW-RTTATTTCGTCTTGCGAATTTCATTAAACG
For amplification of signal peptides
  YolC-FCGCTGCTTGATGTGATCATCCGCGGCATTATGTTTGAATTTCCGTTTAAAGAATGGGCTG
  YolC-RCGGTGAACAGCTCCTCGCCCTTGGATGCTTCGATTAAAGTAATACCTGACATAATCAAAGC
  ywaD-FTCGAATGAGCTTACAGGATCCATGAAAAAGCTTTTGACTGTCATGACG
  ywaD-RTTCTAATTACCCTCCCCCGGGTTATTTGATATCTTCAAAAATGTCAGATGCTTTCGC
), ArticleFig(id=1243300005965054862, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1242175011402903708, language=CN, label=表2, caption=

本研究中所用的引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers nameSequences (5′→3′)
For the construction of expression cassette of SipW
  sipW-Up-FCCCTTAACGAATTCCTGCAGCCCCTGATACGTCAGGCACCGTG
  sipW-Up-RAATCAAGCTGAAGATTACGGCATAC
  sipW-Down-FGTATGCCGTAATCTTCAGCTTGATTCATGAGAGAAACGGGACCATGTC
  sipW-Down-RCACCGCGGTGGCGGCCGCTCTAGTCTTTACCGACTGTCAGAACCG
  sipW-djh-FCCGACTGCGCAAAAGACATAATC
  sipW-djh-RCTGCCTGAGGCAGCATTAAC
  sipW-sjh-FGTGGTGGCGAATACGAATGC
  sipW-sjh-RCTGCCTGAGGCAGCATTAAC
  sipW-FATGGGAGGAAATCACTTGAAATCAG
  sipW-RTTATTTCGTCTTGCGAATTTCATTAAACG
For amplification of signal peptides
  YolC-FCGCTGCTTGATGTGATCATCCGCGGCATTATGTTTGAATTTCCGTTTAAAGAATGGGCTG
  YolC-RCGGTGAACAGCTCCTCGCCCTTGGATGCTTCGATTAAAGTAATACCTGACATAATCAAAGC
  ywaD-FTCGAATGAGCTTACAGGATCCATGAAAAAGCTTTTGACTGTCATGACG
  ywaD-RTTCTAATTACCCTCCCCCGGGTTATTTGATATCTTCAAAAATGTCAGATGCTTTCGC
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解淀粉芽孢杆菌Ⅰ型信号肽酶与信号肽的相互作用
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郭洁洁 , 王伟红 , 何杰民 , 李登科 , 刘业学 , 路福平 , 李玉 *
微生物学报 | 研究报告 2025,65(1): 256-267
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微生物学报 | 研究报告 2025, 65(1): 256-267
解淀粉芽孢杆菌Ⅰ型信号肽酶与信号肽的相互作用
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郭洁洁, 王伟红, 何杰民, 李登科, 刘业学, 路福平, 李玉*
作者信息
  • 天津科技大学 生物工程学院, 工业发酵微生物教育部重点实验室, 天津 300457
Interaction between type Ⅰ signal peptidase and signal peptides in Bacillus amyloliquefaciens
Jiejie GUO, Weihong WANG, Jiemin HE, Dengke LI, Yexue LIU, Fuping LU, Yu LI*
Affiliations
  • Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, School of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China
出版时间: 2025-01-04 doi: 10.13343/j.cnki.wsxb.20240504
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摘要
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【目的】基于Ⅰ型信号肽酶在分泌系统中的重要作用，探究信号肽酶与信号肽之间的作用关系，指导氨肽酶在解淀粉芽孢杆菌中的分泌表达优化。【方法】对解淀粉芽孢杆菌TCCC 19030的内源性信号肽酶和信号肽进行筛选，采用相对荧光强度、酶活力等表征手段进行分析，同时采用分子对接技术探究信号肽酶和信号肽的相互作用关系。【结果】信号肽YolC融合氨肽酶的胞外酶活力最高，达到11 847.67 U/mL。过表达信号肽酶SipW后，氨肽酶的酶活力提高到16 261 U/mL。分子对接结果显示，信号肽YolC与信号肽酶SipW结合自由能最低，为−4.4 kcal/mol。【结论】信号肽优化与信号肽酶过表达可有效提高氨肽酶的分泌表达，其中信号肽酶与信号肽的结合能是影响目的蛋白分泌量的关键因素之一。

信号肽酶  /  信号肽  /  分子对接技术

[Objective] Based on the critical role of type Ⅰ signal peptidase in the secretion system, this study explores the interaction between signal peptidase and signal peptides to guide the optimization of aminopeptidase secretion expression in Bacillus amyloliquefaciens. [Methods] The endogenous signal peptidase and signal peptide of Bacillus amyloliquefaciens TCCC 19030 were examined using relative fluorescence intensity and enzyme activity for analysis, and molecular docking to study their interaction. [Results] The signal peptide YolC fused with aminopeptidase exhibited the highest extracellular enzyme activity, reaching 11 847.67 U/mL. Overexpression of the signal peptidase SipW increased aminopeptidase activity to 16 261 U/mL. Molecular docking results also showed that YolC had the lowest binding free energy with SipW, at −4.4 kcal/mol. [Conclusion] Optimization of signal peptides and overexpression of signal peptidase can effectively enhance the secretion of aminopeptidase. The binding energy between signal peptidase and signal peptide is a key factor influencing the secretion levels of the target protein.

signal peptidase  /  signal peptide  /  molecular docking
郭洁洁, 王伟红, 何杰民, 李登科, 刘业学, 路福平, 李玉. 解淀粉芽孢杆菌Ⅰ型信号肽酶与信号肽的相互作用. 微生物学报, 2025 , 65 (1) : 256 -267 . DOI: 10.13343/j.cnki.wsxb.20240504
Jiejie GUO, Weihong WANG, Jiemin HE, Dengke LI, Yexue LIU, Fuping LU, Yu LI. Interaction between type Ⅰ signal peptidase and signal peptides in Bacillus amyloliquefaciens[J]. Acta Microbiologica Sinica, 2025 , 65 (1) : 256 -267 . DOI: 10.13343/j.cnki.wsxb.20240504
正文
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信号肽是一段连续的氨基酸序列，可协助蛋白质从细胞内跨膜分泌到细胞外[1-2]。在原核生物中，蛋白的分泌几乎离不开信号肽，近年来有不少学者通过优化信号肽进一步提高目标产物的产量，Khadye等[3]对枯草芽孢杆菌(Bacillus subtilis) 168基因组的173个内源信号肽文库进行了高通量筛选，使得耐热β-葡萄糖苷酶分泌量比出发菌株提高了16倍。Shi等[4]通过建立信号肽pelB的突变文库，使得PET水解酶的分泌量提高了1.7倍。然而，当信号肽牵引前体蛋白完成跨膜转运后，信号肽酶可以识别信号肽C端区域的最后3个氨基酸残基，并将其切割，使信号肽与成熟蛋白断开，从而使成熟蛋白分泌至胞外[5]。然而，不同表达水平的信号肽酶显著影响外源蛋白的分泌水平，优化信号肽酶同样是提高目的蛋白表达量的策略之一[6]。王豪[7]在地衣芽孢杆菌中，通过构建信号肽酶基因sipV过表达菌株，使得纳豆激酶酶活达到35.6 FU/mL，相对于对照菌株提高了16%。Cai等[8]以纳豆激酶作为表征蛋白，并敲除了地衣芽孢杆菌的4个Ⅰ型信号肽酶基因sipS、sipT、sipW和sipV，其中sipV的缺失对纳豆激酶活性的影响最大，另外过表达sipV后，纳豆激酶活性比初始菌株提高了4.68倍。Owji等[9]研究指出，信号肽酶的切割效率是蛋白分泌的限速步骤之一。因此，研究信号肽酶与信号肽之间的相互作用，对于提高外源蛋白的高效表达至关重要。尽管大部分细菌中的信号肽酶已经被鉴定[7]，但是关于信号肽酶与信号肽之间的相互作用关系是如何影响外源蛋白分泌的报道相对较少。目前，研究分子间的相互作用常用到分子对接模拟技术，Li等[10]通过分子对接和自由能计算，直观地反映出信号肽酶与信号肽之间的结合程度，随后将sipA整合到解淀粉芽孢杆菌基因组中过量表达，使胞外AprE活性相对于原始菌株提高了19.9%。因此利用对接结果分析信号肽酶与信号肽之间的相互作用，可为提升底盘菌株的分泌效率提供一定的指导和帮助。
解淀粉芽孢杆菌作为重要的工业底盘菌株，其分泌蛋白能力极强，并且参与了多种酶制剂的生产[11]，因此本研究以解淀粉芽孢杆菌(Bacillus amyloliquefaciens) TCCC 19030为出发菌株，以氨肽酶(YwaD)的酶活力为评价指标，筛选出最优信号肽，并以该信号肽为研究对象，利用分子对接、实验验证等方法探究信号肽与信号肽酶之间的作用关系，为进一步提升外源蛋白在解淀粉芽孢杆菌中的分泌表达效率提供参考和依据。
1 材料与方法
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1.1 材料
1.1.1 菌种和质粒
解淀粉芽孢杆菌(B. amyloliquefaciens) TCCC 19030为本实验室保藏，表达载体pLY-3为本实验室前期构建。本研究中使用的菌株、质粒详情信息见表1，所用引物详细信息见表2，引物由天津金唯智生物科技有限公司合成。
1.1.2 酶和试剂
DNA聚合酶、限制性内切核酸酶均购自TaKaRa公司；无缝克隆酶购自北京全式金生物技术有限公司；酶活试剂盒购自Megazyme公司；质粒提取试剂盒、DNA切胶回收试剂盒、DNA抽提试剂盒均购自Omega公司；卡那霉素、溶菌酶等均购自北京索莱宝科技有限公司。
1.1.3 培养基
种子培养基(LB液体培养基)：酵母粉5.0 g/L，蛋白胨10.0 g/L，氯化钠10.0 g/L，溶于自来水中，121 ℃灭菌20 min。
发酵培养基：豆饼粉60.0 g，0.5 mol/L的NaOH溶液溶于1 L去离子水中混匀，pH 12.0，50 ℃下搅拌2 h，待温度冷却至室温之后，加入磷酸直到pH 7.3，13 000 r/min离心10 min后获得上清液，加入0.03% KH2PO4，3%糊精，0.4% Na2HPO4混匀。
1.2 表达载体的构建
以B. subtilis 168基因组为模板克隆出目的基因168AP，并将其构建到pLY-3载体上获得表达载体pLY-3-YwaD。将pLY-3-YwaD和原pLY-3载体分别电转化至B. amyloliquefaciens TCCC 19030，获得重组菌株19030-YwaD和对照菌株19030-NC。
1.3 氨肽酶表达的SDS-PAGE分析
将菌株接种至装有50 mL LB的250 mL摇瓶中，于37 ℃、200 r/min条件下培养48 h，取出发酵液于13 000 r/min离心10 min后获得上清液，随后取出部分上清进行SDS-PAGE分析。浓缩胶浓度5%，电泳电压80 V；分离胶浓度15%，电泳电压120 V。
1.4 融合信号肽表达载体的构建
通过NCBI数据库和SignalP系列软件筛选出20条B. amyloliquefaciens TCCC 19030来源的信号肽序列。随后以B. amyloliquefaciens TCCC 19030的基因组为模板(DNA抽提参考试剂盒)，设计引物PCR扩增信号肽、氨肽酶和pLY-3线性载体的基因序列，采用无缝克隆连接，获得重组质粒，随后将其转化至E. coli JM109感受态中，再将经过甲基化修饰的质粒电转化至B. amyloliquefaciens TCCC 19030，获得重组菌株。
1.5 信号肽酶缺失菌株的构建
采用同源重组的方法敲除4个信号肽酶基因(sipA、sipS、sipW和sipV)，敲除过程以sipW基因为例。使用表1所示的引物sipW-Up-F和sipW-Up-R，以及sipW-Down-F和sipW-Down-R扩增sipW基因同源臂片段，使用BamH I和Xba I双酶切pWH-T2载体获得pWH-T2线性载体。采用无缝克隆技术将sipW基因同源臂片段与pWH-T2线性载体连接，得到重组质粒pWH-T2-SipW。将pWH-T2-SipW转化至E. coli JM109感受态中，再将经过甲基化修饰的重组质粒电转化至B. amyloliquefaciens TCCC 19030，通过Kana抗性筛选出正确的转化子进行单、双交换，双交换验证成功的转化子即为信号肽酶基因sipW敲除成功的菌株。
1.6 信号肽酶sipW过表达菌株的构建
以B. amyloliquefaciens TCCC 19030基因组为模板，使用表1所示引物sipW-F和sipW-R扩增sipW基因片段，使用引物ywaD-F和ywaD-R扩增ywaD基因片段。利用无缝克隆技术将sipW基因片段、ywaD基因片段与pWH-T2线性载体连接，获得重组质粒pWH-SipW-YwaD并转化至E. coli JM109，对转化子进行菌落PCR验证和测序验证，将验证正确的重组质粒转化到E. coli EC135/pM.Bam中进行甲基化修饰，随后电转化至B. amyloliquefaciens TCCC 19030，利用菌落PCR技术验证单、双交换转化子，最终双交换验证成功的转化子即为信号肽酶sipW基因过表达成功的菌株。
1.7 信号肽酶缺失菌株生长曲线的测定
将活化后的菌株挑单菌落接种至5 mL LB液体培养基中，37 ℃、220 r/min振荡培养12 h。以2%的接种量接种至50 mL LB液体培养基中，37 ℃、220 r/min振荡培养6 h后，每隔2 h取样，使用酶标仪在600 nm波长下检测其吸光度值，以时间为横坐标，以OD600值为纵坐标绘制生长曲线，直至检测的OD600值下降，结束取样。
1.8 氨肽酶酶活测定方法
先配制终浓度为40 μg/mL的对硝基苯胺(p-nitroaniline, pNA)标准溶液，以其为母液进行稀释，绘制标准曲线。随后对酶液进行稀释，进行样品的制备和检测。
对照样品制备方法：吸取2.6 mL的Tris-HCl缓冲液加入试管，50 ℃预热5 min，随后加入1 mL 40%的乙酸，反应10 min，再加入0.05 mol/L l-亮氨酸-4-硝基苯胺(l-leucine-p-nitroaniline, LNA) 0.2 mL，混匀后静置10 min。实验样品的制备方法为，吸取0.2 mL酶液，加入含有2.6 mL Tris-HCl缓冲液的试管中，50 ℃预热5 min，加0.05 mol/L的LNA 0.2 mL到试管中，反应10 min，加入1 mL 40%的乙酸终止反应，混匀后静置10 min。最后将对照样品和实验样品在405 nm的条件下检测。
在50 ℃和pH 8.0条件下，每分钟水解亮氨酸对硝基苯胺生成1 μg pNA，即定义为一个酶活单位，以U/mL表示。酶活按照公式(1)计算。
式中：X为由标准曲线得出的样品最终稀释液的酶活力，n为稀释倍数，0.2为加入反应体系的酶液体积，10为反应时间10 min。
1.9 AutoDock分子对接方法
使用AutoDock软件将信号肽末端3个氨基酸与信号肽酶蛋白分子进行模拟对接，具体操作步骤如下。
首先准备受体和配体的.pdb文件。之后用AutoDockTools打开protein.pdb和ligand.pdb，分别输出为protein.pdbqt和ligand. pdbqt形式。接下来对受体蛋白设置对接的盒子大小，盒子要将活性位点完全包裹，保存protein_ligand.gpf文件作为对接的文件。随后，选择protein.pdbqt文件，将受体蛋白质设置为刚性，对接算法选择拉马克遗传算法，保存成为protein_ligand.dpf文件。运行AutoGrid和AutoDock程序进行对接，对接的结果文件保存为protein_ligand.dlg文件。最后利用PyMOL进行配体与受体的可视化对接。
2 结果与分析
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2.1 氨肽酶的分泌表达
将重组菌19030-YwaD和对照菌19030-NC分别以1%接种量接种至装50 mL LB的250 mL摇瓶中，于37 ℃、200 r/min条件下培养48 h。发酵液13 000 r/min离心10 min后取上清液进行SDS-PAGE分析，结果如图1所示。与对照相比，在约49.00 kDa大小的位置出现了一条蛋白条带，与氨肽酶相对分子量理论值49.45 kDa大小一致。此外，观察到对照组在49.50 kDa左右也存在略浅条带，这是因为B. amyloliquefaciens TCCC 19030胞外蛋白组中的蛋白种类众多，不排除存在与氨肽酶分子量大小相似的条带。随后，取发酵上清液进行氨肽酶酶活检测，结果表明重组菌19030-YwaD氨肽酶酶活为6 056 U/mL，而对照菌19030-NC未检测到酶活。通过检测酶活以及SDS-PAGE条带深浅的双重分析，可以证明枯草芽孢杆菌来源的氨肽酶在B. amyloliquefaciens TCCC 19030成功分泌表达。
2.2 信号肽的筛选
通过基因组数据和SignalP系列软件分析并鉴定出20条B. amyloliquefaciens TCCC 19030来源的信号肽序列，选用氨肽酶的酶活力作为信号肽评价指标。将20个重组菌株分别以1%接种量接种至250 mL摇瓶中，每个摇瓶装50 mL LB培养基，于37 ℃、220 r/min条件下培养48 h，每个重组菌株设置3个平行，发酵结果如图2所示。酶活相对较高的5个分别是氨肽酶融合了YolC、YdbK、YqzG、BglC和Lytb信号肽，相比于对照菌株分别提高了34.19%、25.01%、27.07%、25.42%和25.17%，其中信号肽YolC融合氨肽酶时，氨肽酶酶活最高，达到11 847.67 U/mL，最终选取最优信号肽YolC进行后续实验。
2.3 信号肽酶基因缺失菌株的构建及其对菌体生长的影响
不同信号肽酶的表达影响外源蛋白的分泌水平。利用生物信息学工具对B. amyloliquefaciens TCCC 19030基因组进行了系统的比对分析，最终确定基因组上存在这4种Ⅰ型信号肽酶，分别是信号肽酶SipA、SipS、SipW和SipV，其在基因组上的相对位置如图3所示，大小分别为581、557、584和515 bp。采用同源重组法缺失4个Ⅰ型信号肽酶基因sipA、sipS、sipW和sipV，4个信号肽酶基因缺失成功的菌株分别命名为19030-ΔA、19030-ΔS、19030-ΔW和19030-ΔV，接下来探究这4种Ⅰ型信号肽酶缺失对相关信号肽以及外源蛋白分泌的影响。
据报道，信号肽酶的缺失不仅影响蛋白质的分泌，一定程度上也影响细胞的生长[12-13]。因此，对4株敲除菌株19030-ΔA、19030-ΔV、19030-ΔS和19030-ΔW进行生长曲线测定，结果如图4所示，进入对数生长期后，19030-ΔW菌株的OD600值略高于对照菌株TCCC 19030，但差异不显著。其余3株敲除菌株与对照菌株TCCC 19030相比生长曲线无明显差异，表明Ⅰ型信号肽酶的缺失对菌株的生长不会造成显著影响。Cai等[8]缺失了地衣芽孢杆菌的4个信号肽基因sipS、sipT、sipV和sipW后并未导致细菌生长停滞，本研究结果与该报道一致。
2.4 Ⅰ型信号肽酶的缺失对氨肽酶分泌的影响
将pLY-3-YolC-YwaD分别电转化至4种Ⅰ型信号肽酶敲除菌株和原始菌株中，分别命名为BY-ΔA、BY-ΔV、BY-ΔS、BY-ΔW和BY-19030。上述电转化成功的5个重组菌株分别以1%接种量接种至装50 mL LB的250 mL摇瓶中，于37 ℃、220 r/min条件下培养48 h，每个重组菌株设置3个平行，发酵结果如图5所示。信号肽酶缺失菌株的氨肽酶酶活均低于对照菌株，其中信号肽酶基因sipW缺失的菌株BY-ΔW，氨肽酶酶活力最低，在48 h的酶活力仅为对照菌株BY-19030的67.36%，说明在解淀粉芽胞杆菌中信号肽酶SipW对氨肽酶的分泌表达影响最大。信号肽酶基因sipA、sipS、sipV的缺失对氨肽酶分泌表达的影响较小。后续实验对4种信号肽酶进行过表达验证，以确定关键信号肽酶的功能。
2.5 Ⅰ型信号肽酶过表达对氨肽酶分泌的影响
为进一步探究4种Ⅰ型信号肽酶对氨肽酶分泌的影响。利用基因组整合的方法将信号肽酶基因sipA、sipS、sipW和sipV进行过表达，并将质粒pLY-3-YolC-YwaD分别电转化至4种Ⅰ型信号肽酶过表达菌株中，重组菌株分别命名为19030-YwaD-SipA、19030-YwaD-SipS、19030-YwaD-SipW、19030-YwaD-SipV。以BY-19030为对照菌株，将4种重组菌株和BY-19030分别以1%接种量接种至装50 mL LB的250 mL摇瓶中，于37 ℃、220 r/min条件下培养48 h，每个重组菌株设置3个平行，发酵结果如图6所示。
在12、24、36和48 h测得的菌株19030-YwaD-SipW酶活力均高于对照菌株，发酵培养48 h，氨肽酶酶活达到最高，为16 261 U/mL，比对照菌株高27.75%。然而在信号肽酶基因sipA、sipS和sipV过表达的菌株中，氨肽酶酶活却无明显差异。
随后将4种信号肽酶基因过表达菌株的发酵上清液进行SDS-PAGE，结果如图7所示，信号肽酶基因sipW的过表达对氨肽酶在解淀粉芽胞杆菌中的分泌表达影响最大，而信号肽酶基因sipA、sipS和sipV过表达对氨肽酶的分泌无显著性影响，SDS-PAGE结果与酶活测定结果一致，表明信号肽酶基因sipW的过表达可以通过提高氨肽酶的分泌表达量，进而提高酶活。
2.6 信号肽与信号肽酶之间的作用分析
研究表明，在4种信号肽酶中只有信号肽酶SipW对氨肽酶的分泌有明显的促进作用。为了探究造成这种差异的原因，采用分子对接技术模拟4种信号肽酶与信号肽YolC之间的相互作用。选取信号肽YolC末端的3个氨基酸(脯氨酸-天冬酰胺-赖氨酸，P-N-K)作为配体，采用PyMOL软件建立三肽模型。此外，利用网站SWISS-MODEL (https://swissmodel.expasy.org/interactive)预测了信号肽酶SipA、SipS、SipW和SipV的结构作为受体(图8)。
使用AutoDock软件将信号肽YolC与上述4种信号肽酶进行分子对接[14]，对接得到的4个模型如图9所示。通过观察配体与受体相互作用的结构示意图，能够清晰地看到配体在受体结合口袋中的空间位置和相互作用情况，在分子半柔性对接的最优构象中，YolC末端的3个氨基酸建立的三肽模型以不同方式插入4种信号肽酶受体结合口袋中。此外，在结构示意图中可以明显看到配体与受体的特定氨基酸残基之间存在相互作用力，这使得配体稳定存在于结合口袋中，因此对接结果以结合自由能为最终评价，结合自由能越低，构象趋于稳定，表明对接的结果越好[15]。如图10所示，信号肽YolC与信号肽酶SipA、SipS、SipV和SipW的对接结合自由能分别为–2.8、–3.5、–3.5和–4.4 kcal/mol。其中信号肽YolC与SipW的结合自由能最低，说明信号肽酶SipW与信号肽YolC的结合能力最强，信号肽酶SipW行使切割功能的可能性越大，越有利于外源蛋白的分泌[13]，这与氨肽酶酶活测定结果一致。
3 讨论与结论
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解淀粉芽孢杆菌在洗涤、皮革、食品和医药等行业中应用广泛，是原核表达系统中分泌表达外源蛋白的理想宿主。很多研究在解淀粉芽孢杆菌菌株的改造、表达元件以及发酵条件优化等方面都做了大量的工作以提升目标产物的产量。Zhang等[16]通过串联缺失肽聚糖水解酶lytD、lytE和sigD基因，使耐酸α-淀粉酶酶活力提升了48.4%。张莹等[17]通过生物信息学手段预测及筛选启动子，获得新型启动子，提高了解淀粉芽孢杆菌碱性果胶酶的表达量。邝嘉华等[18]通过单因素试验和响应面试验对解淀粉芽孢杆菌BSK532的发酵条件进行了优化，使得其胞外多糖产量达到171.31 mg/L，较原始条件提高155.7%。然而，由于解淀粉芽孢杆菌分泌系统的复杂性[19]，提高分泌蛋白的产量仍然是一个挑战。大多数研究人员也通过优化信号肽以及信号肽酶来提升目的蛋白的产量，王茂军等[20]通过对5种信号肽进行筛选和组合，重组菌株的果聚糖蔗糖酶酶活力达到125.76 U/mL，较原始酶活力提高了100.49%。蒋蕊[21]筛选出引导α-淀粉酶高效分泌的信号肽yoml，使得酶活达到113.25 U/mL。
本研究以解淀粉芽孢杆菌TCCC 19030为出发菌株，构建分泌表达载体，筛选出氨肽酶分泌的最优信号肽YolC，相比于对照菌株，氨肽酶酶活提高了34.19%。然而效果好的信号肽只能将更多的前体蛋白引导到膜上，必须经过信号肽酶的切割才能促进蛋白质的成熟。在此基础上，本研究进一步考察了信号肽酶对氨肽酶分泌的影响，4种信号肽酶SipA、SipS、SipV和SipW都会对氨肽酶的分泌产生影响，其中信号肽酶SipW对氨肽酶分泌的影响最大。然而通过过表达关键信号肽酶SipW使氨肽酶酶活提高了27.75%。此外，Malten等[6]通过在巨大芽胞杆菌中过表达SPaseI型基因sipM，使葡聚糖蔗糖酶的产量提高了3.7倍。
然而，如何快速、合理地筛选信号肽，确定关键的信号肽酶，以及研究两者之间的相互作用关系，对于提高分泌效率至关重要。随着计算工具的迅猛发展，分子模拟对接技术日趋成熟。任绍东[22]从信号肽引导分泌功能的角度出发，基于信号肽的三维结构，利用分子对接的方法设计了信号肽的共通H区氨基酸序列，获得了能够与原始信号肽分泌能力相媲美的信号肽。吴怡[23]通过分子对接、体内外活性等一系列筛选实验，最终获得3个具有P. aeruginosa群体感应系统抑制作用的噁唑烷酮类化合物：MM11、MM16及MM22。其中MM11浓度在200 μmol/L时，可减少约30%胞外蛋白酶分泌。本研究利用生物信息学软件AutoDocK、PyMOL和SWISS-MODEL，将信号肽YolC与4种Ⅰ型信号肽酶进行模拟对接，其中信号肽酶SipW与信号肽YolC的结合自由能最低，说明信号肽酶SipW与信号肽YolC的结合能力最强，行使切割功能的可能性最大，有利于外源蛋白的分泌。证实了将关键信号肽酶SipW进行过表达是提高目的蛋白分泌效率的有效手段之一，本研究为其他芽孢杆菌分泌系统的优化提供了参考。
基金
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  • 国家重点研发计划(2021YFC2104000)
文献
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参考文献 引证文献
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2025年第65卷第1期
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doi: 10.13343/j.cnki.wsxb.20240504
  • 接收时间：2024-08-15
  • 首发时间：2026-03-21
  • 出版时间：2025-01-04
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  • 收稿日期：2024-08-15
  • 录用日期：2024-10-30
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National Key Research and Development Program of China(2021YFC2104000)
国家重点研发计划(2021YFC2104000)
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    天津科技大学 生物工程学院, 工业发酵微生物教育部重点实验室, 天津 300457

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2种不同金属材料的力学参数
科
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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