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Article(id=1241376211142767155, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20230720, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1700928000000, receivedDateStr=2023-11-26, revisedDate=null, revisedDateStr=null, acceptedDate=1708272000000, acceptedDateStr=2024-02-19, onlineDate=1773896752391, onlineDateStr=2026-03-19, pubDate=1714752000000, pubDateStr=2024-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773896752391, onlineIssueDateStr=2026-03-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773896752391, creator=13701087609, updateTime=1773896752391, updator=13701087609, issue=Issue{id=1241376204247331313, tenantId=1146029695717560320, journalId=1192105938417971205, year='2024', volume='64', issue='5', pageStart='1331', pageEnd='1682', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1773896750747, creator=13701087609, updateTime=1773897643611, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241379949253284790, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241379949253284791, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241376204247331313, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1538, endPage=1549, ext={EN=ArticleExt(id=1241376214431101561, articleId=1241376211142767155, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Phosphate-responsive promoter region inHeyndrickxia coagulans, columnId=1241045257748533520, journalTitle=Acta Microbiologica Sinica, columnName=Research Articles, runingTitle=null, highlight=null, articleAbstract=

Heyndrickxia coagulans characterized by low nutrient requirements, high titers of fermentation products, and thermal tolerance has become a major microbial species for lactic acid fermentation. We have demonstrated that phosphate stimulates the gene expression of L-lactate dehydrogenase inH.coagulans to increase L-lactic acid production, the mechanism of which, however, remains unknown. [Objective] To primarily investigated the mechanism of phosphate in stimulating the gene transcription of lactate dehydrogenase inH.coagulans. [Methods] RT-PCR was employed to analyze the transcriptional level changes of lactate dehydrogenase inH.coagulans after phosphate addition. The core promotor region responsive to phosphate stimulation was identified by conventional methods of molecular biology. [Results] The key element responsive to phosphate was located in the upstream promoter region of the L-lactate dehydrogenase gene. The core region of the promoter was responsible for phosphate stimulation. The identified promoter was employed to promote D-lactate production with diammonium phosphate addition. [Conclusion] This study reported a new phosphate-responsive gene element, providing a theoretical basis for improving the synthesis efficiency of other biochemicals.

, correspAuthors=Bo YU, authorNote=null, correspAuthorsNote=
*YU Bo, 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=Xiaoxiao QI, Limin WANG, Bo YU), CN=ArticleExt(id=1241376216033325830, articleId=1241376211142767155, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=凝结芽孢杆菌磷酸盐响应启动子的研究, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

耐热凝结芽孢杆菌因其对营养要求简单、发酵产物浓度高以及耐高温等特点，已成为乳酸发酵的主要菌种。在前期的研究中，我们发现磷酸盐可以激活凝结芽孢杆菌L-乳酸脱氢酶基因的转录，从而提高乳酸产量。然而，磷酸盐如何激活乳酸脱氢酶的基因表达，目前还不清楚，也未有类似的研究报道。【目的】对凝结芽孢杆菌响应磷酸盐的调控机制进行研究。【方法】通过RT-PCR分析磷酸盐添加时凝结芽孢杆菌乳酸脱氢酶转录水平变化，确定响应磷酸盐的关键元件区域，进一步通过分子生物学手段，分析凝结芽孢杆菌响应磷酸盐的关键基因片段。【结果】确定了响应磷酸盐的关键元件位于乳酸脱氢酶基因上游启动子区，解析了响应磷酸盐的L-乳酸脱氢酶启动子核心区，利用该启动子及核心区能够有效驱动外源D-乳酸脱氢酶基因的表达，实现在凝结芽孢杆菌中D-乳酸的合成。【结论】本研究有望获得一种新的响应磷酸盐的调控元件，为提高其他生物化学品的合成效率改造提供参考。

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MARTÍNEZ-CASTRO M.Regulation of the phosphate metabolism inStreptomyces genus: impact on the secondary metabolites[J].Applied Microbiology and Biotechnology,2019,103(4):1643-1658., articleTitle=Regulation of the phosphate metabolism inStreptomyces genus: impact on the secondary metabolites, refAbstract=null), Reference(id=1241446132446974780, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, doi=null, pmid=null, pmcid=null, year=2015, volume=6, issue=null, pageStart=402, pageEnd=null, url=null, language=null, rfNumber=[21], rfOrder=21, authorNames=null, journalName=Frontiers in Microbiology, refType=null, unstructuredReference=SANTOS-BENEIT F.The Pho regulon: a huge regulatory network in bacteria[J].Frontiers in Microbiology,2015,6:402., articleTitle=The Pho regulon: a huge regulatory network in bacteria, refAbstract=null), Reference(id=1241446132593775431, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, doi=10.1128/AEM.01864-14, pmid=null, pmcid=null, year=2014, volume=80, issue=23, pageStart=7134, pageEnd=7141, url=null, language=null, rfNumber=[22], rfOrder=22, authorNames=null, journalName=Applied and Environmental Microbiology, refType=null, unstructuredReference=WANG LM, CAI YM, ZHU LF, GUO HL, YU B.Major role of NAD-dependent lactate dehydrogenases in the production of L-lactic acid with high optical purity by the thermophileBacillus coagulans[J].Applied and Environmental Microbiology,2014,80(23):7134-7141., articleTitle=Major role of NAD-dependent lactate dehydrogenases in the production of L-lactic acid with high optical purity by the thermophileBacillus coagulans, refAbstract=null), Reference(id=1241446132732187466, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, doi=null, pmid=null, pmcid=null, year=2009, volume=75, issue=11, pageStart=3528, pageEnd=3534, url=null, language=null, rfNumber=[23], rfOrder=23, authorNames=null, journalName=Applied and Environmental Microbiology, refType=null, unstructuredReference=AHN J, HONG J, PARK M, LEE H, LEE E, KIM C, LEE J, CHOI ES, JUNG JK, LEE H.Phosphate-responsive promoter of aPichia pastoris sodium phosphate symporter[J].Applied and Environmental Microbiology,2009,75(11):3528-3534., articleTitle=Phosphate-responsive promoter of aPichia pastoris sodium phosphate symporter, refAbstract=null)], funds=[Fund(id=1241446125459264123, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, awardId=31970025, language=EN, fundingSource=National Natural Science Foundation of China(31970025), fundOrder=null, country=null), Fund(id=1241446127120208512, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, awardId=31970025, language=CN, fundingSource=国家自然科学基金(31970025), fundOrder=null, country=null), Fund(id=1241446127220871812, tenantId=1146029695717560320, 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ext=[AuthorCompanyExt(id=1241446118802903382, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, companyId=1241446118790320469, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 University of Chinese Academy of Sciences, Beijing 100049, China), AuthorCompanyExt(id=1241446118807097687, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, companyId=1241446118790320469, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 中国科学院大学, 北京 100049)])], figs=[ArticleFig(id=1241446122753937905, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 1, caption=The concentration of L-lactic acid during 24 h fermentation ofHeyndrickxia coagulans 36D1 in different media. Cl: The inorganic salt medium with NH4Cl; H: The inorganic salt medium with (NH4)2HPO4. ****:P < 0.000 1. Data were shown as the mean of three replicates, with the error bars representing±standard error., figureFileSmall=r1EIvYu8qK8scHMLzUZQkQ==, figureFileBig=S1rEAkifHnyILIkRUv411Q==, tableContent=null), ArticleFig(id=1241446122900738551, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图1, caption=凝结芽孢杆菌36D1在不同培养基中发酵24 h的L-乳酸浓度, figureFileSmall=r1EIvYu8qK8scHMLzUZQkQ==, figureFileBig=S1rEAkifHnyILIkRUv411Q==, tableContent=null), ArticleFig(id=1241446123051733506, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 2, caption=The verification of strainHeyndrickxia coagulans DSM1ΔldhL1ΔldhL2ΔPDSM1-1182. Lane A: The PCR amplification product of strain with PDSM1-1182 deletion; Lane B: The PCR amplification product of strainH.coagulans DSM1ΔldhL1ΔldhL2., figureFileSmall=wAjP+RJYOBbl+XiimJ7EZw==, figureFileBig=sZxTkOuBNyMDAkkrSfRTPg==, tableContent=null), ArticleFig(id=1241446123198534152, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图2, caption=凝结芽孢杆菌DSM1ΔldhL1ΔldhL2ΔPDSM1-1182菌株验证, figureFileSmall=wAjP+RJYOBbl+XiimJ7EZw==, figureFileBig=sZxTkOuBNyMDAkkrSfRTPg==, tableContent=null), ArticleFig(id=1241446123320168977, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 3, caption=The transfer efficiencies under different affinity time. Data were shown as the mean of three replicates, with the error bars representing±standard error., figureFileSmall=4PT9Trrk1w2YBRP2cCwVPQ==, figureFileBig=EsZVoyhQj34uQKQ69PncwA==, tableContent=null), ArticleFig(id=1241446123425026585, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图3, caption=不同亲和时间条件的质粒转化效率, figureFileSmall=4PT9Trrk1w2YBRP2cCwVPQ==, figureFileBig=EsZVoyhQj34uQKQ69PncwA==, tableContent=null), ArticleFig(id=1241446123555050020, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 4, caption=Identification of the core region ofldhL promoter ofHeyndrickxia coagulans 36D1. A: The positions of different promoters in P1182. B: The signal strength of different promoters represented by sfGFP. Data were shown as the mean of three replicates, with the error bars representing±standard error., figureFileSmall=LuF538PuA/ECDMwkJL8wLQ==, figureFileBig=XeSf7PO4ph0SJOXxrUkHXQ==, tableContent=null), ArticleFig(id=1241446123672490541, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图4, caption=凝结芽孢杆菌36D1乳酸脱氢酶基因ldhL上游P1182内启动子核心区, figureFileSmall=LuF538PuA/ECDMwkJL8wLQ==, figureFileBig=XeSf7PO4ph0SJOXxrUkHXQ==, tableContent=null), ArticleFig(id=1241446123806708275, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 5, caption=L-lactic acid production andldhL gene transcription levels driven by different promoter. A: L-lactic acid synthesis level per unit bacterial volume. B: The transcription level ofldhL was increased in fermentation medium containing (NH4)2HPO4 compared with NH4Cl fermentation medium. Data were shown as the mean of three replicates, with the error bars representing±standard error., figureFileSmall=5f3pS3/b1fpD4xHuArtIoQ==, figureFileBig=6WdRdbRoQqExNLgU2BXfsA==, tableContent=null), ArticleFig(id=1241446123936731706, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图5, caption=不同启动子驱动下的L-乳酸产量及ldhL基因转录水平, figureFileSmall=5f3pS3/b1fpD4xHuArtIoQ==, figureFileBig=6WdRdbRoQqExNLgU2BXfsA==, tableContent=null), ArticleFig(id=1241446124091920962, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Figure 6, caption=The variation ofldhD transcription and D-lactate production under different concentrations of diammonium phosphate. A: The multiplier of increased transcription levels ofldhD in each expression plasmid regulated by (NH4)2HPO4 compared with NH4Cl. P1182: The plasmid named pNW33N-TraJ-36D1-P1182+ldhD; PG: The plasmid named pNW33N-TraJ-36D1-PG+ldhD. B: D-lactic acid production under different concentration (NH4)2HPO4 culture conditions. Data were shown as the mean of three replicates, with the error bars representing±standard error., figureFileSmall=b6iAtT5NvsV7Mr1rnqBQFw==, figureFileBig=WNJXWcyeTrjAKbuUyVGfPg==, tableContent=null), ArticleFig(id=1241446124184195654, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=图6, caption=不同表达质粒的宿主菌中ldhD转录水平和在不同浓度(NH4)2HPO4培养条件下 D-乳酸产量, figureFileSmall=b6iAtT5NvsV7Mr1rnqBQFw==, figureFileBig=WNJXWcyeTrjAKbuUyVGfPg==, tableContent=null), ArticleFig(id=1241446124301636170, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Table 1, caption=

The strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株名称
Strain name		用途
Usage		来源
Source
Escherichia coli S17-1The donor strain for conjugation transferKeep in our laboratory
Heyndrickxia coagulans DSM1The wild strainKeep in our laboratory
Heyndrickxia coagulans 36D1The wild strainKeep in our laboratory
DSM1ΔldhL1ΔldhL2Two lactate dehydrogenase genes were knocked out in DSM1[12]
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182The experimental chassis strain was further knocked out the promoterConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PG+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PG+ldhLConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PG+ldhDDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PG+ldhDConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PL+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PL+ldhLConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-P1182+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-P1182+ldhLConstructed in this study
E.coli Top10-36D1-P1182+sfGFPE.coli Top10 with pRG-36D1-P1182+sfGFPConstructed in this study
E.coli Top10-36D1-PG+sfGFPE.coli Top10 with pRG-36D1-PG+sfGFPConstructed in this study
E.coli Top10-36D1-PL+sfGFPE.coli Top10 with pRG-36D1-PL+sfGFPConstructed in this study
E.coli Top10-36D1-PJ+sfGFPE.coli Top10 with pRG-36D1-PJ+sfGFPConstructed in this study
E.coli Top10-36D1-PI+sfGFPE.coli Top10 with pRG-36D1-PI+sfGFPConstructed in this study
), ArticleFig(id=1241446124431659601, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=表1, caption=

  本研究使用的菌株

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株名称
Strain name		用途
Usage		来源
Source
Escherichia coli S17-1The donor strain for conjugation transferKeep in our laboratory
Heyndrickxia coagulans DSM1The wild strainKeep in our laboratory
Heyndrickxia coagulans 36D1The wild strainKeep in our laboratory
DSM1ΔldhL1ΔldhL2Two lactate dehydrogenase genes were knocked out in DSM1[12]
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182The experimental chassis strain was further knocked out the promoterConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PG+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PG+ldhLConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PG+ldhDDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PG+ldhDConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-PL+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-PL+ldhLConstructed in this study
DSM1ΔldhL1ΔldhL2ΔPDSM1-1182-36D1-P1182+ldhLDSM1ΔldhL1ΔldhL2ΔPDSM1-1182 with pNW33N-TraJ-36D1-P1182+ldhLConstructed in this study
E.coli Top10-36D1-P1182+sfGFPE.coli Top10 with pRG-36D1-P1182+sfGFPConstructed in this study
E.coli Top10-36D1-PG+sfGFPE.coli Top10 with pRG-36D1-PG+sfGFPConstructed in this study
E.coli Top10-36D1-PL+sfGFPE.coli Top10 with pRG-36D1-PL+sfGFPConstructed in this study
E.coli Top10-36D1-PJ+sfGFPE.coli Top10 with pRG-36D1-PJ+sfGFPConstructed in this study
E.coli Top10-36D1-PI+sfGFPE.coli Top10 with pRG-36D1-PI+sfGFPConstructed in this study
), ArticleFig(id=1241446124553294423, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Table 2, caption=

The plasmids used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
质粒名称
Plasmid name		用途
Usage		来源
Source
pNW33NPlasmid, CmR[7]
pNW33N-TraJPlasmid for conjugation transfer, CmRConstructed in this study
pNW33N-TraJ-36D1-PG+ldhLldhL was expressed with PG as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-PG+ldhDldhD was expressed with PG as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-PL+ldhLldhL was expressed with PL as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-P1182+ldhLldhL was expressed with P1182 as promoter, CmRConstructed in this study
pRG-36D1-P1182+sfGFPsfGFP was expressed with P1182 as promoter, AmpRConstructed in this study
pRG-36D1-PG+sfGFPsfGFP was expressed with PG as promoter, AmpRConstructed in this study
pRG-36D1-PL+sfGFPsfGFP was expressed with PL as promoter, AmpRConstructed in this study
pRG-36D1-PB+sfGFPsfGFP was expressed with PB as promoter, AmpRConstructed in this study
pRG-36D1-PE+sfGFPsfGFP was expressed with PE as promoter, AmpRConstructed in this study
pRG-36D1-PJ+sfGFPsfGFP was expressed with PJ as promoter, AmpRConstructed in this study
pRG-36D1-PI+sfGFPsfGFP was expressed with PI as promoter, AmpRConstructed in this study
pMH77Knockout vector, CmR[7]
pMH77-PDSM1-1182The vector to knockout PDSM1-1182, CmRConstructed in this study
), ArticleFig(id=1241446124658152024, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=表2, caption=

  本研究使用的质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
质粒名称
Plasmid name		用途
Usage		来源
Source
pNW33NPlasmid, CmR[7]
pNW33N-TraJPlasmid for conjugation transfer, CmRConstructed in this study
pNW33N-TraJ-36D1-PG+ldhLldhL was expressed with PG as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-PG+ldhDldhD was expressed with PG as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-PL+ldhLldhL was expressed with PL as promoter, CmRConstructed in this study
pNW33N-TraJ-36D1-P1182+ldhLldhL was expressed with P1182 as promoter, CmRConstructed in this study
pRG-36D1-P1182+sfGFPsfGFP was expressed with P1182 as promoter, AmpRConstructed in this study
pRG-36D1-PG+sfGFPsfGFP was expressed with PG as promoter, AmpRConstructed in this study
pRG-36D1-PL+sfGFPsfGFP was expressed with PL as promoter, AmpRConstructed in this study
pRG-36D1-PB+sfGFPsfGFP was expressed with PB as promoter, AmpRConstructed in this study
pRG-36D1-PE+sfGFPsfGFP was expressed with PE as promoter, AmpRConstructed in this study
pRG-36D1-PJ+sfGFPsfGFP was expressed with PJ as promoter, AmpRConstructed in this study
pRG-36D1-PI+sfGFPsfGFP was expressed with PI as promoter, AmpRConstructed in this study
pMH77Knockout vector, CmR[7]
pMH77-PDSM1-1182The vector to knockout PDSM1-1182, CmRConstructed in this study
), ArticleFig(id=1241446124867867231, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Table 3, caption=

Ct values ofldhL inHeyndrickxia coagulans determined by RT-PCR under different cultivation conditions

, figureFileSmall=null, figureFileBig=null, tableContent=
SampleHousekeeping gene (16S rRNA gene)Gene of interest (ldhL)2–ΔΔCt
NH4Cl8.64±0.1026.55±0.1113.78±0.78
(NH4)2HPO49.06±0.0623.18±0.08
), ArticleFig(id=1241446124985307748, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=表3, caption=

  凝结芽孢杆菌乳酸脱氢酶基因ldhL在不同培养条件下的Ct数值

, figureFileSmall=null, figureFileBig=null, tableContent=
SampleHousekeeping gene (16S rRNA gene)Gene of interest (ldhL)2–ΔΔCt
NH4Cl8.64±0.1026.55±0.1113.78±0.78
(NH4)2HPO49.06±0.0623.18±0.08
), ArticleFig(id=1241446125169857131, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=EN, label=Table 4, caption=

Ct values ofldhL in engineered strain were determined by RT-PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
SampleHousekeeping gene (16S rRNA gene)Gene of interest (ldhL)2–ΔΔCt
NH4Cl9.03±0.0925.71±0.055.31±0.20
(NH4)2HPO48.79±0.1023.07±0.05
), ArticleFig(id=1241446125253743218, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241376211142767155, language=CN, label=表4, caption=

  工程菌株中ldhL基因RT-PCR测定Ct数值

, figureFileSmall=null, figureFileBig=null, tableContent=
SampleHousekeeping gene (16S rRNA gene)Gene of interest (ldhL)2–ΔΔCt
NH4Cl9.03±0.0925.71±0.055.31±0.20
(NH4)2HPO48.79±0.1023.07±0.05
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凝结芽孢杆菌磷酸盐响应启动子的研究
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祁肖肖 1, 2 , 王丽敏 1 , 于波 1, *
微生物学报 | 研究报告 2024,64(5): 1538-1549
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微生物学报 | 研究报告 2024, 64(5): 1538-1549
凝结芽孢杆菌磷酸盐响应启动子的研究
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祁肖肖1, 2, 王丽敏1, 于波1, *
作者信息
  • 1 中国科学院微生物研究所微生物生理与代谢工程研究室, 北京 100101
  • 2 中国科学院大学, 北京 100049
Phosphate-responsive promoter region inHeyndrickxia coagulans
Xiaoxiao QI1, 2, Limin WANG1, Bo YU1, *
Affiliations
  • 1 Department of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • 2 University of Chinese Academy of Sciences, Beijing 100049, China
出版时间: 2024-05-04 doi: 10.13343/j.cnki.wsxb.20230720
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耐热凝结芽孢杆菌因其对营养要求简单、发酵产物浓度高以及耐高温等特点，已成为乳酸发酵的主要菌种。在前期的研究中，我们发现磷酸盐可以激活凝结芽孢杆菌L-乳酸脱氢酶基因的转录，从而提高乳酸产量。然而，磷酸盐如何激活乳酸脱氢酶的基因表达，目前还不清楚，也未有类似的研究报道。【目的】对凝结芽孢杆菌响应磷酸盐的调控机制进行研究。【方法】通过RT-PCR分析磷酸盐添加时凝结芽孢杆菌乳酸脱氢酶转录水平变化，确定响应磷酸盐的关键元件区域，进一步通过分子生物学手段，分析凝结芽孢杆菌响应磷酸盐的关键基因片段。【结果】确定了响应磷酸盐的关键元件位于乳酸脱氢酶基因上游启动子区，解析了响应磷酸盐的L-乳酸脱氢酶启动子核心区，利用该启动子及核心区能够有效驱动外源D-乳酸脱氢酶基因的表达，实现在凝结芽孢杆菌中D-乳酸的合成。【结论】本研究有望获得一种新的响应磷酸盐的调控元件，为提高其他生物化学品的合成效率改造提供参考。

凝结芽孢杆菌  /  磷酸盐响应  /  启动子  /  乳酸脱氢酶

Heyndrickxia coagulans characterized by low nutrient requirements, high titers of fermentation products, and thermal tolerance has become a major microbial species for lactic acid fermentation. We have demonstrated that phosphate stimulates the gene expression of L-lactate dehydrogenase inH.coagulans to increase L-lactic acid production, the mechanism of which, however, remains unknown. [Objective] To primarily investigated the mechanism of phosphate in stimulating the gene transcription of lactate dehydrogenase inH.coagulans. [Methods] RT-PCR was employed to analyze the transcriptional level changes of lactate dehydrogenase inH.coagulans after phosphate addition. The core promotor region responsive to phosphate stimulation was identified by conventional methods of molecular biology. [Results] The key element responsive to phosphate was located in the upstream promoter region of the L-lactate dehydrogenase gene. The core region of the promoter was responsible for phosphate stimulation. The identified promoter was employed to promote D-lactate production with diammonium phosphate addition. [Conclusion] This study reported a new phosphate-responsive gene element, providing a theoretical basis for improving the synthesis efficiency of other biochemicals.

Heyndrickxia coagulans  /  phosphate-responsive  /  promoter  /  lactate dehydrogenase
祁肖肖, 王丽敏, 于波. 凝结芽孢杆菌磷酸盐响应启动子的研究. 微生物学报, 2024 , 64 (5) : 1538 -1549 . DOI: 10.13343/j.cnki.wsxb.20230720
Xiaoxiao QI, Limin WANG, Bo YU. Phosphate-responsive promoter region inHeyndrickxia coagulans[J]. Acta Microbiologica Sinica, 2024 , 64 (5) : 1538 -1549 . DOI: 10.13343/j.cnki.wsxb.20230720
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凝结芽孢杆菌(Heyndrickxia coagulans)最初是从腐败的炼乳中分离获得的，属于厚壁菌门，是一种革兰氏阳性菌，其最适生长温度为45−50 ℃，最适生长pH值为6.6−7.0[1]。凝结芽孢杆菌通过糖酵解途径将葡萄糖转化为丙酮酸，进而通过乳酸脱氢酶催化生成乳酸[2]。与常温乳杆菌相比，凝结芽孢杆菌具有诸多优势，该菌对培养基成分要求比较简单，发酵温度相对较高，在50−60 ℃均可快速繁殖生长，因此不易受到杂菌污染，可进行开放式发酵，而且该菌能够以较快的速度生产高光学纯度的L-乳酸，是一种非常理想的工业生产菌株[3-4]。
目前，关于凝结芽孢杆菌的遗传操作研究相对较少，仅有少量文献报道了在凝结芽孢杆菌中可自主复制的质粒，但是该菌的电转化效率和基因敲除效率均较低[5]。Rhee等从凝结芽孢杆菌P4-102B中提取质粒pMSR0，并以其为骨架，构建了能在凝结芽孢杆菌和大肠杆菌中表达的穿梭质粒pMSR10，并成功通过电转化转入凝结芽孢杆菌P4-102B，但并不能在其他凝结芽孢杆菌中重复该结果[6]。Kovács等利用Cre-lox系统敲除了凝结芽孢杆菌DSM1中的ldhL和sigF基因[7]。Wang等通过基因敲除技术，获得了凝结芽孢杆菌QZ19ΔldhLΔals突变株，但是敲除效率只有1:5 000[8]。目前，仅有DSM1、QZ19及P4-102B这3株凝结芽孢杆菌菌株被报道可以实现遗传操作，但其遗传操作系统还需进一步改进。受制于遗传操作，目前对凝结芽孢杆菌的研究主要集中在L-乳酸的发酵工艺上，对代谢机理解析方面的研究较少。
我们前期发现(NH4)2HPO4具有激活凝结芽孢杆菌L-乳酸脱氢酶基因转录的功能，从而提高乳酸发酵水平，而其他包括各种铵盐类的无机氮源并无上述激活功能[9]。在该过程中，起主要作用的因素是磷酸盐，而非铵根离子[9]。通过广泛的文献检索，截至目前，仅乳酸乳杆菌(Lactobacillus lactis) RM2-24中有报道过类似现象[10]。Singhvi等报道添加(NH4)2HPO4可以激活菌株RM2-24的 D-乳酸脱氢酶的基因表达[10]。在后续的研究中，Singhvi等进一步发现，相较于原始菌株L.lactis NCIM2368，突变株RM2-24的L-乳酸脱氢酶基因表达可以被(NH4)2HPO4激活，而其他无机氮源则无法产生相同效果，然而作者并未深入解析相关机制[11]。目前，关于凝结芽孢杆菌对磷酸盐响应机制的研究尚处于空白状态。本研究对凝结芽孢杆菌36D1菌株L-乳酸脱氢酶基因上游启动子区域开展了分析，尝试解析凝结芽孢杆菌中可响应磷酸盐激活并提高L-乳酸脱氢酶基因转录水平的关键区域。
1 材料与方法
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1.1 材料
1.1.1 菌株和质粒
Escherichia coli Top10购自北京擎科生物科技股份有限公司。野生型菌株Heyndrickxiacoagulans 36D1和H.coagulans DSM1 (GenBank登录号：CP009709.1)由本实验室保藏。L-乳酸脱氢酶双基因敲除菌株H.coagulans DSM1ΔldhL1ΔldhL2由本课题组前期构建，详见文献[12]。表达载体pNW33N和基因敲除载体pMH77由本实验室保藏，其质粒信息详见文献[7]。本研究使用的菌株和质粒信息详见表1和表2。
1.1.2 培养基
乳酸发酵513培养基(g/L)：葡萄糖50.0，酵母粉10.0，CaCO3 30.0，115 ℃灭菌20 min。
无机盐发酵培养基H (g/L)：葡萄糖60.0，酵母粉0.9，(NH4)2HPO4 3.0，甜菜碱0.2，CaCO3 72.0，115 ℃灭菌20 min。
对照无机盐发酵培养基Cl (g/L)：葡萄糖60.0，酵母粉0.9，NH4Cl 2.43，甜菜碱0.2，CaCO3 72.0，115 ℃灭菌20 min。
BC培养基(g/L)：蔗糖50.0，酵母粉10.0，(NH4)2HPO4 2.0，(NH4)2SO4 3.5，Bis-Tris 10.0，pH 6.6−6.7，115 ℃灭菌20 min。培养基在使用之前按量加入过滤除菌的微量元素浓缩混合液，各微量元素的终浓度及种类(g/L)：CaCl2 0.003，MgCl2 0.005，CoCl2·6H2O 0.2×10−3，CuCl2·2H2O 0.1×10−4，H3BO3 0.3×10−3，Na2MoO4·2H2O 0.3×10−4，NiSO4·6H2O 0.2×10−4，MnCl2·4H2O 0.3×10−4，ZnCl₂ 0.5×10−4，固体培养基灭菌后加1 g/L MgCl2。
RG培养基：BC培养基中加入17.11 g/L蔗糖，1.1 g/L葡萄糖，pH 6.6−6.7，115 ℃灭菌20 min，使用之前加入终浓度分别为0.41 g/L的MgCl2和0.01 g/L的CaCl2。
1.2 凝结芽孢杆菌基因敲除
采用同源重组方法敲除凝结芽孢杆菌DSM1菌株L-乳酸脱氢酶基因上游1 182 bp基因。首先将含有待敲除基因同源臂的敲除载体pMH77-PDSM1-1182电转化入DSM1ΔldhL1ΔldhL2菌种中，挑选转化菌株接入含7 μg/mL氯霉素BC液体培养基中，45 ℃、120 r/min培养12 h。然后，将菌株依照10%的接种量接入新的含7 μg/mL氯霉素的BC液体培养基中，45 ℃、120 r/min连续传3代；将第3代的菌株在45 ℃、120 r/min培养12 h后再放入60 ℃、120 r/min培养12 h；取菌液涂布于含有氯霉素抗性的平板，55 ℃培养20 h，筛选发生第一次同源重组的菌株；挑取发生一次同源重组的菌株接入无抗的BC培养基中，连续传代后涂布于不含抗生素的BC平板上，筛选基因敲除菌株。并通过PCR验证和测序正确后，获得凝结芽孢杆菌1 182敲除菌株。
1.3 基因转录测定
野生菌株H.coagulans 36D1在513培养基(100 mL摇瓶中加入50 mL培养基)中50 ℃、120 r/min培养12 h并连续活化3代，分别接种于无机盐发酵培养基H、对照无机盐发酵培养基Cl使其初始OD600达到0.35，然后在50 ℃、120 r/min条件下培养5−6 h。含有表达质粒的工程菌在含有7 μg/mL氯霉素的513培养基(100 mL摇瓶中加入50 mL培养基)中50 ℃、120 r/min活化3代，分别接种于含有7 μg/mL氯霉素的发酵培养基H和对照发酵培养基Cl，初始OD600值为0.2，50 ℃、120 r/min条件下培养7−9 h，收集细胞提取总RNA。使用E.Z.N.A.细菌RNA试剂盒(Omega公司)提取总RNA。总RNA浓度通过260 nm处的吸光度测定。参考FastQuant RT Kit (With gDNase) [天根生化科技(北京)有限公司]的方法合成cDNA拷贝，使用SYBR Cycler 96 RT-PCR检测系统(SYBR PremixEx Taq, TaKaRa公司)扩增。将2 μL cDNA作为模板，加入20 μL实时PCR混合物，加入10 pmol的基因特异性引物，测定不同cDNA浓度的每个PCR的阈值周期(Ct)，并与内参DNA (16S rRNA基因)的阈值周期进行比较。采用2–ΔΔCt相对定量法测定mRNA水平。每组数据重复4次，结果为不同cDNA样品的相对表达量。
1.4 乳酸含量的测定
使用HPLC测定发酵液中乳酸的含量，色谱柱为有机酸柱(Aminex HPX-87H；Bio-Rad公司)。流动相为6 mmol/L H2SO4，流速为0.5 mL/min，柱温55 ℃，进样体积为10 μL。
1.5 接合转移质粒构建及转化
使用来源于穿梭质粒pKV12 (GenBank登录号：JQ679011.1)的TraJ蛋白基因构建接合转移质粒[13-14]。采用Gibson Assembly将TraJ蛋白基因插入大肠杆菌-芽孢杆菌穿梭载体pNW33N的大肠杆菌复制起点ori和repB之间，构建接合转移重组质粒pNW33N-TraJ。将构建好的pNW33N-TraJ质粒转入大肠杆菌S17-1中，LB培养基37 ℃、200 r/min活化含有pNW33N-TraJ质粒的S17-1菌株；使用含有10 g/L葡萄糖的BC培养基50 ℃、120 r/min活化凝结芽孢杆菌DSM1；取活化好的两菌株在37 ℃亲和反应12 h；刮取亲和后的菌体，重悬于BC培养基中，并进行10−1、10−2和10−3梯度稀释；将稀释的菌液涂布于含有7 μg/mL氯霉素的BC平板，50 ℃静置培养15 h。
2 结果与分析
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2.1 (NH4)2HPO4提高凝结芽孢杆菌36D1中L-乳酸脱氢酶基因ldhL转录水平验证
前期研究发现，在培养基中添加(NH4)2HPO4可以显著上调凝结芽孢杆菌2-6菌株的L-乳酸脱氢酶基因的转录水平，进而提高L-乳酸产量[9]。本研究进一步验证了凝结芽孢杆菌36D1菌株是否存在相同的现象。虽然凝结芽孢杆菌36D1基因组上同时含有L-乳酸脱氢酶LdhL和 D-乳酸脱氢酶LdhD，但两个基因距离位置较远且启动子不同，并且36D1菌株的 D-乳酸产量极低，因此，本文排除了对 D-乳酸脱氢酶LdhD的研究，聚焦L-乳酸脱氢酶基因ldhL的研究。以含有NH4Cl的无机盐发酵培养基Cl为对照，观测菌株在含有(NH4)2HPO4的无机盐发酵培养基H中的ldhL的转录水平。RT-PCR测定数值如表3所示。与NH4Cl培养条件相比，在含有(NH4)2HPO4的培养基中凝结芽孢杆菌36D1中的ldhL的转录水平提高了13.78倍。进一步测定上述2个培养条件下，L-乳酸的产量。如图1所示，(NH4)2HPO4培养条件下，L-乳酸的产量达到(56.75±0.52) g/L，比NH4Cl培养条件下的(9.12±0.19) g/L提升了6倍。因此，本研究再次确认(NH4)2HPO4可以提高凝结芽孢杆菌菌株的L-乳酸脱氢酶基因转录水平。
2.2 凝结芽孢杆菌DSM1ΔldhL1ΔldhL2 ΔPDSM1-1182菌株构建
上述研究结果显示(NH4)2HPO4可以提高凝结芽孢杆菌36D1的ldhL转录水平。因此，在凝结芽孢杆菌ldhL的启动子区域可能存在响应(NH4)2HPO4的调控区域。依据Kovács等的研究，凝结芽孢杆菌L-乳酸脱氢酶上游1 182 bp的区域含有乳酸脱氢酶启动子(GenBank登录号：GU323916.1)[7]。因此，本研究聚焦在ldhL上游启动子区域(以下简称为P1182)。由于目前还无法对凝结芽孢杆菌36D1进行遗传转化等基因操作，本研究选取可进行遗传操作的凝结芽孢杆菌DSM1菌株为宿主，开展相关的研究。为排除DSM1菌株自身ldhL上游启动子区域的干扰，我们首先利用文献报道的基于温敏质粒pMH77的基因敲除系统[7]，敲除DSM1菌株的L-乳酸脱氢酶基因上游1 182 bp的片段(记为PDSM1-1182)。构建凝结芽孢杆菌ldhL上游1 182 bp敲除质粒pMH77-PDSM1-1182，转化L-乳酸脱氢酶双基因敲除菌株DSM1ΔldhL1ΔldhL2。PCR验证及基因测序证实敲除该基因片段。如图2所示，电泳结果证实PDSM1-1182片段的敲除成功，进一步基因测序证实成功构建了不含有自身ldhL上游启动子区的菌株，命名为H.coagulans DSM1ΔldhL1ΔldhL2ΔPDSM1-1182菌株，并用于后续的研究中。
2.3 凝结芽孢杆菌结合转移质粒构建
虽然凝结芽孢杆菌DSM1是可以进行遗传操作的菌株，但其电转化效率较低，尤其对大于7 kb质粒的电转化效率极低，成为该菌遗传操作的限制因素。接合转移法常被用于地衣芽孢杆菌、枯草芽孢杆菌等菌株的外源基因转化，本研究尝试建立凝结芽孢杆菌接合转移质粒，用于满足后续的分子操作需求。以大肠杆菌S17-1作为供体菌，选择pNW33N-TraJ质粒为研究对象，优化供体菌与受体菌的亲和时间的实验结果，如图3显示，亲和时间12 h的条件下，获得最高的转化效率，最终确定两菌株最佳亲和时间为12 h。当质粒大小为7 000 bp时，接合转移的转化效率可达到4.6×10−4 transconjugants/recipient。本研究也是首次报道在凝结芽孢杆菌中采用结合转移方法实现质粒转化。
2.4 凝结芽孢杆菌36D1应答(NH4)2HPO4的启动子区域鉴定
2.4.1 凝结芽孢杆菌36D1的ldhL上游1 182 bp基因功能验证
前述实验数据证实(NH4)2HPO4可以提高凝结芽孢杆菌ldhL的转录水平，首先测试了响应磷酸盐的调控关键位点是否位于ldhL上游启动子区内。通过克隆36D1菌株完整的L-乳酸脱氢酶基因和上游P1182启动子序列，构建了重组质粒pNW33N-TraJ-36D1-1182+ldhL，并将该重组质粒导入H.coagulans DSM1ΔldhL1ΔldhL2ΔPDSM1-1182菌株，测试乳酸脱氢酶对磷酸盐的响应情况。表达菌株在含有氯霉素的513培养基中进行活化，随后分别接种至仅以NH4Cl为无机氮来源的发酵培养基Cl和仅以(NH4)2HPO4为无机氮来源的发酵培养基H中，培养至指数期，收集菌体提取总RNA，测定L-乳酸脱氢酶的表达情况。RT-PCR结果显示(表4)，与NH4Cl培养菌体相比，在重组菌株中，(NH4)2HPO4的存在使ldhL的转录水平提高了5.31倍，表明凝结芽孢杆菌36D1来源的ldhL上游的1 182 bp区域确实存在可以响应(NH4)2HPO4的位点。
2.4.2 凝结芽孢杆菌36D1的ldhL上游响应磷酸盐启动子核心区域的确定
P1182区域内存在参与(NH4)2HPO4调控的基因元件，但是具体的区域还需进一步研究。首先在大肠杆菌系统中开展核心功能区域的实验。通过启动子预测网站(www.denovodna.com)对ldhL上游的P1182片段进行预测，发现P1182序列中含有多个启动子序列，如图4A所示，将ldhL上游的P1182片段分为PB、PE、PL、PJ、PG和PI这6个预测的可能启动子区域。这些启动子区域均具有可能的−35区、−10区及启动子序列。以绿色荧光蛋白sfGFP为表征信号[15]，分别构建质粒pRG-36D1-PL+sfGFP、pRG-36D1-PB+sfGFP、pRG-36D1-PE+sfGFP、pRG-36D1-PJ+sfGFP、pRG-36D1-PG+sfGFP和pRG-36D1-PI+sfGFP，以全长片段pRG-36D1-P1182+sfGFP质粒作为阳性对照，将上述所构建的质粒转化大肠杆菌TOP10菌株，培养至指数中后期进行取样检测荧光信号强度。图4B结果显示PL、PB、PE、PG具有明显的启动子效应，PB可分为PE与PI两部分，PI无明显的启动子效应，PE可分为PJ与PG两部分，PJ无明显的启动子效应，由此推断PG为凝结芽孢杆菌36D1的ldhL的核心启动子。
2.4.3 凝结芽孢杆菌36D1中响应(NH4)2HPO4的核心启动子PG验证
在大肠杆菌中以绿色荧光蛋白sfGFP表征获得核心启动子区域后，进一步在凝结芽孢杆菌中以乳酸脱氢酶ldhL为表达基因进行重复验证。使用上文初步鉴定的PL和PG启动子分别驱动ldhL基因表达，构建pNW33N-TraJ-36D1-PL+ldhL、pNW33N-TraJ-36D1-PG+ldhL表达质粒，以全基因片段P1182构建的表达质粒pNW33N-TraJ-36D1- P1182+ldhL为对照。将构建的表达质粒接合转移H.coagulans DSM1ΔldhL1ΔldhL2ΔPDSM1-1182菌株中，进而在乳酸发酵513培养基中测定每单位OD600的L-乳酸合成水平。如图5A所示，PL和PG启动子均可以驱动ldhL基因的表达从而合成乳酸，但PG的启动子活性更强，所产的乳酸含量最高。与大肠杆菌数据基本一致，PG启动子的活性甚至高于完整P1182的启动子活性，PL也有一定的启动子活性，但相比之下，产生了最低量的L-乳酸，进一步确认PG为ldhL的核心启动子。
为验证PG启动子是否是响应磷酸盐的核心启动子，进一步检测了在(NH4)2HPO4添加条件下，重组菌株中ldhL的转录水平。将含有表达质粒pNW33N-TraJ-36D1-PL+ldhL、pNW33N-TraJ-36D1- PG+ldhL和pNW33N-TraJ-36D1-P1182+ldhL的菌株接入含有氯霉素抗性的513培养基中进行活化，将活化好的菌株分别接入含有NH4Cl的无机盐发酵培养基与含有(NH4)2HPO4的无机盐发酵培养基中培养至指数期，取样收集菌体提取总RNA。以在含有NH4Cl的无机盐发酵培养基中ldhL的转录水平为对照，计算菌株在含有(NH4)2HPO4的无机盐发酵培养基中ldhL的转录水平的变化情况。如图5B所示，在(NH4)2HPO4发酵培养基中，含有PG启动子和完整P1182的菌株表现出ldhL转录水平明显上调，其中PG启动子驱动的菌株ldhL转录水平上调倍数达到15倍。上述实验结果进一步明确PG启动子是凝结芽孢杆菌响应磷酸盐的核心启动子区域。值得注意的是，PL启动子也表现出一定的对磷酸盐响应的现象，但上调倍数要小于完整P1182驱动的数据，其具体功能还需后续进一步的研究。
2.5 (NH4)2HPO4响应启动子PG在 D-乳酸合成菌株的应用
Singhvi等报道，添加(NH4)2HPO4可以激活乳酸乳球菌RM2-24的 D-乳酸脱氢酶的基因表达[10]。为了验证PG启动子在磷酸盐条件下对异源基因的作用，选取来源于德氏乳杆菌的 D-乳酸脱氢酶为例[16]，以全长P1182启动子驱动 D-乳酸脱氢酶基因ldhD作为对照，构建了重组表达质粒pNW33N-TraJ-36D1-PG+ldhD和pNW33N-TraJ-36D1-P1182+ldhD，将上述2个表达质粒接合转移转入菌株H.coagulans DSM1ΔldhL1ΔldhL2ΔPDSM1-1182，构建的重组菌株在含有氯霉素抗性的乳酸发酵513培养基中进行培养。将菌株分别接入含有NH4Cl的无机盐发酵培养基与含有(NH4)2HPO4的无机盐发酵培养基，培养至指数期，取样收集菌体提取总RNA。如图6A所示，在(NH4)2HPO4培养条件下，启动子PG和P1182都可以上调 D-乳酸脱氢酶的转录水平，并且PG启动子的活性更高。这说明除L-乳酸脱氢酶基因的启动子，对 D-乳酸脱氢酶也具有同样的激活效果。本研究进一步测定了在不同(NH4)2HPO4添加浓度和PG启动子驱动下，重组菌株产 D-乳酸的情况。如图6B所示，随着(NH4)2HPO4添加量的增加， D-乳酸的产量呈现出相应的上升趋势，在7 g/L (NH4)2HPO4添加量时获得 D-乳酸的最高产量，但进一步提高(NH4)2HPO4添加量到9 g/L时，则造成 D-乳酸合成水平的下降。综上所述，启动子PG在(NH4)2HPO4的激活下也可以提高 D-乳酸脱氢酶的转录水平，进一步确认PG为凝结芽孢杆菌36D1响应磷酸盐核心区域。
3 讨论与结论
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无机氮源(NH4)2SO4和(NH4)2HPO4等被广泛应用于工业发酵过程中[17]。本文作者在前期研究中发现，在培养基中添加(NH4)2HPO4可以提高乳杆菌的乳酸脱氢酶的转录水平，进而提高乳酸的产量，而(NH4)2SO4等无上述功能[9-11]。文献报道，在含有(NH4)2HPO4的培养基中，乳酸乳杆菌RM2-24菌株的乳酸脱氢酶的活力为0.25 U/mg，在不含有(NH4)2HPO4的条件下，其乳酸脱氢酶的活力仅为0.15 U/mg，但磷酸盐如何激活乳酸脱氢酶的表达机制未见报道[10]。无机磷作为六大重要元素，除参与遗传物质的代谢外，还参与许多重要的细胞过程，包括能量代谢和细胞内信号传导等。目前响应无机磷的主要机制是Pho调节子，其构成细菌中非常有效和灵敏的调节机制。它由双组分调节系统控制，包括内膜组氨酸激酶传感蛋白(PhoR)和细胞质转录反应调节因子，其在大肠杆菌中命名为PhoB，在枯草芽孢杆菌中命名为PhoP[18]。在已报道的磷酸盐调节机制中，当无机磷稀缺时，PhoR磷酸化PhoB，磷酸化的PhoB能够结合DNA上的特定序列并激活或抑制靶基因的转录，这些特定序列，称为PHO盒。在大多数细菌中，PHO盒是由7个保守的核苷酸和4个保守性较低的核苷酸组成的11个核苷酸的序列[19]。PHO盒也被广泛发现存在于与无机磷代谢不直接相关的其他基因中，显示Pho调节子的丰富多样性作用，其不仅作为无机磷同化的调节因子，而且可作为细胞代谢过程中的重要调节因子(master regulator)，包括通过蛋白质磷酸化来调节细胞代谢过程[20]。越来越多的研究证实，Pho调节子也参与营养代谢网络(nutritional cross-talk)、次级代谢产物合成以及其他重要细胞过程的调节[21]。乳酸生产是细胞重要的初级代谢途径[22]，乳酸脱氢酶则是胞内乳酸生产的关键酶。本研究中，在添加3 g/L (NH4)2HPO4的条件下，乳酸脱氢酶的转录水平是NH4Cl培养基中的13.78倍。Ahn等在Pichia pastoris中改造获得了磷酸盐响应启动子PHO89[23]，发现启动子直接影响着基因的表达，该启动子可以响应低浓度磷，并启动目的基因的表达。通过分析PHO89启动子序列，得到2个PHO调节系统可能的结合位点。在凝结芽孢杆菌L-乳酸脱氢酶上游区域并未发现类似的PHO盒的序列，显示该菌存在与P.pastoris不同的调控机制。前面通过对乳酸脱氢酶上游1 182 bp的启动子区域进行分析，并结合实验验证，确定了PG为核心启动子。下一步对磷酸盐结合区域的研究将会有助于解析磷酸盐响应机制，凝结芽孢杆菌响应磷酸盐提高L-乳酸脱氢酶基因转录的启动子PG是否与磷酸盐结合的位点相关，后续将会对PG位点进行深入研究，如以PG作为探针捕获与其相互作用的蛋白，通过EMSA及BLI等体外分析技术确定与PG结合的核心蛋白，获得体内参与(NH4)2HPO4对ldhL的转录调控的关键调控因子，将有助于我们进一步解析凝结芽孢杆菌响应磷酸盐的机制。此外，在凝结芽孢杆菌中L-乳酸脱氢酶参与糖酵解、丙酮酸代谢等多个生理过程，本研究发现磷酸盐提高乳酸产量，也可能是磷酸盐的添加增强了丙酮酸的合成水平，从而提高了L-乳酸的产量。因此，磷酸盐对于凝结芽孢杆菌的生理代谢过程的影响机制还有待进一步探究。另一方面，无机盐向胞内的输送是以无机离子形式穿膜。由于磷酸根在水溶液中会存在多种离子形式，依据目前实验数据，我们无法确认胞内激活L-乳酸脱氢酶基因转录的具体磷酸根离子形式。然而，(NH4)2HPO4是工业发酵培养中广泛使用的无机盐，本研究发现并证实凝结芽孢杆菌PG启动子具有响应(NH4)2HPO4的现象，其可以作为工业发酵菌株改造的一个低成本的启动子元件，具有较好的应用潜力。
基金
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  • 国家自然科学基金(31970025)
  • 国家自然科学基金(32070026)
  • 内蒙古自治区“揭榜挂帅”项目(2022JBGS0007)
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2024年第64卷第5期
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doi: 10.13343/j.cnki.wsxb.20230720
  • 接收时间：2023-11-26
  • 首发时间：2026-03-19
  • 出版时间：2024-05-04
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  • 收稿日期：2023-11-26
  • 录用日期：2024-02-19
基金
National Natural Science Foundation of China(31970025)
国家自然科学基金(31970025)
National Natural Science Foundation of China(32070026)
国家自然科学基金(32070026)
Inner Mongolia Autonomous Region Research Project of China(2022JBGS0007)
内蒙古自治区“揭榜挂帅”项目(2022JBGS0007)
作者信息
    1 中国科学院微生物研究所微生物生理与代谢工程研究室, 北京 100101
    2 中国科学院大学, 北京 100049

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species		 占总种数比例
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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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