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Article(id=1226554101106913370, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226554095926952065, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240844, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1735228800000, receivedDateStr=2024-12-27, revisedDate=null, revisedDateStr=null, acceptedDate=1737907200000, acceptedDateStr=2025-01-27, onlineDate=1770362885977, onlineDateStr=2026-02-06, pubDate=1751558400000, pubDateStr=2025-07-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770362885977, onlineIssueDateStr=2026-02-06, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770362885977, creator=13701087609, updateTime=1770362885977, updator=13701087609, issue=Issue{id=1226554095926952065, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='7', pageStart='2771', pageEnd='3233', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770362884741, creator=13701087609, updateTime=1770363575040, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226556991309529548, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226554095926952065, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226556991309529549, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226554095926952065, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3056, endPage=3074, ext={EN=ArticleExt(id=1226554101874471054, articleId=1226554101106913370, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Metabolic engineering of Sphingomonas paucimobilis for the production of gellan gum, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] Using Sphingomonas paucimobilis as the starting strain, a high-yield gellan gum-producing engineered strain was constructed through metabolic engineering, and fermentation process optimization was performed, providing both theoretical support and technical foundations for the efficient biosynthesis of gellan gum with this bacterium. [Methods] A CRISPR-Cas9-based gene editing system was developed for S. paucimobilis, and subsequently employed to genomically integrate two key gellan gum biosynthesis genes: the regulatory protein gene (gelA) and the β-1,4-glucuronosyltransferase gene (gelK), both under the control of constitutive promoters. Building upon this foundation, fermentation parameters including carbon source, nitrogen source, pH, and dissolved oxygen were systematically optimized through single-factor experiments. [Results] The engineered strain FMME-GG08 achieved a gellan gum yield of 10.8 g/L in shake-flask cultivation, representing a 130.2% enhancement over the parental strain. Following fermentation process optimization, the production level reached 20.1 g/L in 15 L scale bioreactors, with a sucrose conversion efficiency of 0.50 g/g. [Conclusion] This study not only successfully constructed a high-yield gellan gum-producing strain and established an efficient fermentation process, providing a reliable technical solution for industrial production, but also developed a genetic editing strategy that serves as an important reference for metabolic engineering of non-model microorganisms to produce other high-value exopolysaccharides.

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*E-mail: LI Xiaomin,
LIU Liming,
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【目的】 以少动鞘氨醇单胞菌为出发菌株，通过代谢工程改造构建高产结冷胶工程菌株，并优化发酵工艺，为利用该菌高效合成结冷胶提供理论支撑与工艺基础。 【方法】 基于CRISPR-Cas9系统开发适用于少动鞘氨醇单胞菌的基因编辑工具，并利用该工具在基因组中整合组成型启动子驱动的结冷胶合成基因簇调控蛋白基因(gelA)和β-1,4-葡萄糖醛酸转移酶基因(gelK)。在此基础上，通过单因素试验优化了碳源、氮源、pH及溶氧等发酵工艺参数。 【结果】 工程菌株FMME-GG08在摇瓶培养中结冷胶产量达到10.8 g/L，较出发菌株提升130.2%。发酵工艺优化后，在15 L规模发酵罐中结冷胶产量达到20.1 g/L，蔗糖转化效率为0.50 g/g。 【结论】 本研究不仅成功构建了结冷胶高产菌株并建立了高效发酵工艺，为工业化生产提供了可靠技术方案，同时所开发的基因编辑策略也为代谢工程改造少动鞘氨醇单胞菌等非模式微生物生产其他高价值胞外多糖提供了重要参考。

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作者贡献声明

孙鹏：研究构思和设计、实验操作、论文撰写与修改；薛正莲：发酵工艺放大指导；高聪：基因编辑工具构建指导；刘佳：发酵培养基优化指导；吴静：发酵工艺优化指导；李晓敏：研究设计、实验指导、论文指导与修改；刘立明：研究设计、论文指导与修改。

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Advances in the biosynthesis of gellan gum by Sphingomonas paucimobilis [J]. 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A: Colony count statistics of different plasmid origins of replication on plates; B: Copy number of different plasmids in S. paucimobilis strains., figureFileSmall=f77dKVWAUpcs8z8lKeyMjQ==, figureFileBig=XEeibHmQDWcWZ37K8qiCIg==, tableContent=null), ArticleFig(id=1227681735442366524, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图3, caption=不同来源质粒复制子的验证。A：不同复制子平板菌落数统计；B：不同质粒在少动鞘氨醇单胞菌菌株中的拷贝数。, figureFileSmall=f77dKVWAUpcs8z8lKeyMjQ==, figureFileBig=XEeibHmQDWcWZ37K8qiCIg==, tableContent=null), ArticleFig(id=1227681735526252609, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 4, caption=Characterization of promoters strength. A: Construction of strongly composed promoter expression plasmids from different sources; B: Fluorescence/OD600 of strain fermented in flask for 24 h., figureFileSmall=AXto7JFnefCHBArt7N+uUA==, figureFileBig=9zorlmpgF2s58q1Y968OYQ==, tableContent=null), ArticleFig(id=1227681735618527299, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图4, caption=启动子强度表征。A：不同来源强组成型启动子表达质粒的构建；B：摇瓶发酵24 h菌株的荧光/OD600。, figureFileSmall=AXto7JFnefCHBArt7N+uUA==, figureFileBig=9zorlmpgF2s58q1Y968OYQ==, tableContent=null), ArticleFig(id=1227681735689830472, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 5, caption=Construction of Sphingomonas paucimobilis strain gene editing system. A: Figure of the pLO3-eGFP recombinant plasmid of the triparental conjugation system and the mobilizable plasmid pRK2013; B: INTEGRATE system plasmid map; C: CRISPR-Cpf1 system plasmid map; D: RecET-Cre/loxP system plasmid map and target gene integration frame; E: CRISPR-Cas9 system plasmid map and target gene integration frame., figureFileSmall=p0XcO80PTlmdt1/oCN9hhQ==, figureFileBig=bLEZtIRjR/33F6h5MK/RKA==, tableContent=null), ArticleFig(id=1227681735752745038, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图5, caption=少动鞘氨醇单胞菌基因编辑系统构建。A：三亲接合系统的pLO3-eGFP重组质粒和游动质粒pRK2013质粒图；B：INTEGRATE系统质粒图；C：CRISPR-Cpf1系统质粒图；D：RecET-Cre/loxP系统质粒图和目的基因整合框；E：CRISPR-Cas9系统质粒图和目的基因整合框。, figureFileSmall=p0XcO80PTlmdt1/oCN9hhQ==, figureFileBig=bLEZtIRjR/33F6h5MK/RKA==, tableContent=null), ArticleFig(id=1227681735853408338, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 6, caption=Verification of gene editing systems. A: Fluorescent colorimetric plate for different gene integration systems; B: CRISPR-Cas9 plate fluorescent strain PCR validation electrophoresis map and mutant strain sequencing map; C: INTEGRATE plate fluorescent strain PCR validation electrophoresis map and mutant strain sequencing map., figureFileSmall=T9mMlYyiIxmnxWS2MBOmgg==, figureFileBig=EzexKiDW9BMLKi1XOL54uw==, tableContent=null), ArticleFig(id=1227681735954071640, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图6, caption=基因编辑系统验证。A：不同基因整合系统荧光显色平板；B：CRISPR-Cas9平板荧光菌株PCR验证电泳图和突变菌株测序图；C：INTEGRATE平板荧光菌株PCR验证电泳图和突变菌株测序图。, figureFileSmall=T9mMlYyiIxmnxWS2MBOmgg==, figureFileBig=EzexKiDW9BMLKi1XOL54uw==, tableContent=null), ArticleFig(id=1227681736029569118, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 7, caption=Shaken bottle verification of recombinant strains., figureFileSmall=CNSKuSNUdrZMXD6E7zDq4g==, figureFileBig=fatJfwpIXaLaq2d2X8XflA==, tableContent=null), ArticleFig(id=1227681736155398247, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图7, caption=重组菌株的摇瓶验证, figureFileSmall=CNSKuSNUdrZMXD6E7zDq4g==, figureFileBig=fatJfwpIXaLaq2d2X8XflA==, tableContent=null), ArticleFig(id=1227681736298004590, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 8, caption=Fermentation indexes of FMME-GG08 with flasks. A: Comparison of gelA transcription level between the recombinant strain FMME-GG08 and the wild-type strain FMME-GG01; B: Comparison of gelK transcription level between the recombinant strain FMME-GG08 and the wild-type strain FMME-GG01; C: Cell dry weight curve during shake flask fermentation; D: Yield of gellan gum during shake flask fermentation., figureFileSmall=x0Zc///D0YphMmLBYaVbPg==, figureFileBig=C+7reZxxUBAgQk4k0Ffnkw==, tableContent=null), ArticleFig(id=1227681736419639412, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图8, caption=FMME-GG08摇瓶发酵参数。A：gelA基因转录水平比较；B：gelK基因转录水平比较；C：摇瓶发酵过程中菌体细胞干重曲线；D：摇瓶发酵过程中结冷胶产量。, figureFileSmall=x0Zc///D0YphMmLBYaVbPg==, figureFileBig=C+7reZxxUBAgQk4k0Ffnkw==, tableContent=null), ArticleFig(id=1227681736541274234, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 9, caption=Shake flask growth curves of strain FMME-GG08. A: Growth curves of S. paucimobilis primary seed culture in shake flask; B: Growth curves of S. paucimobilis secondary shake flask seed culture., figureFileSmall=x+hOPM6FNKgbF+6sKyRn5A==, figureFileBig=7tzuf47FneAiK1BwoRJP0A==, tableContent=null), ArticleFig(id=1227681736667103361, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图9, caption=FMME-GG08菌株种子摇瓶生长曲线。A：少动鞘氨醇单胞菌一级种子摇瓶生长曲线；B：少动鞘氨醇单胞菌二级种子摇瓶生长曲线。, figureFileSmall=x+hOPM6FNKgbF+6sKyRn5A==, figureFileBig=7tzuf47FneAiK1BwoRJP0A==, tableContent=null), ArticleFig(id=1227681736784543879, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Figure 10, caption=Fermentation process optimization. A: Bar chart of fermentation using glucose, sucrose, and maltose syrup as the sole carbon source; B: Bar chart of fermentation with different sugar feeding strategies; C: Bar chart of fermentation using peptone, soybean meal powder, corn steep liquor, and wheat bran extract as organic nitrogen sources; D: Bar chart of fermentation with different concentrations of organic nitrogen sources; E: Bar chart of fermentation with different pH control strategies; F: Bar chart of fermentation with different dissolved oxygen control strategies; G: Fermentation curves of the strain in a 15 L fermenter under optimal fermentation conditions., figureFileSmall=wvhmmogL5VBPBeHOmG4YXQ==, figureFileBig=OyyafXPBDmSQgHA6x1Gzzw==, tableContent=null), ArticleFig(id=1227681736901984397, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=图10, caption=发酵工艺优化。A：分别选取葡萄糖、蔗糖、麦芽糖浆为唯一碳源的发酵柱状图；B：不同补糖策略的发酵柱状图；C：分别选取蛋白胨、豆粕粉、玉米浆和麸皮提取物为有机氮源的发酵柱状图；D：不同浓度有机氮源的发酵柱状图；E：不同pH调控策略的发酵柱状图；F：不同溶氧调控策略的发酵柱状图；G：最优发酵工艺下菌株在15 L发酵罐中的发酵曲线。, figureFileSmall=wvhmmogL5VBPBeHOmG4YXQ==, figureFileBig=OyyafXPBDmSQgHA6x1Gzzw==, tableContent=null), ArticleFig(id=1227681737027813525, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Table 1, caption=

Strains and plasmids used in this study

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NamesRelevant characteristicsSources
Strains
E. coli JM109E. coli JM109 wild-type strainLab stock
E. coli S17-1E. coli S17-1 carrying pLO3Lab stock
E. coli HB101E. coli HB101 carrying pRK2013Lab stock
FMME-GG01Wild-type Sphingomonas paucimobilisLab stock
FMME-GG02FMME-GG01 carrying pBBR1-pgmThis study
FMME-GG03FMME-GG01 carrying pBBR1-vgbThis study
FMME-GG04FMME-GG01 carrying pBBR1-gelAThis study
FMME-GG05FMME-GG01 carrying pBBR1-gelKThis study
FMME-GG06FMME-GG01 carrying pBBR1-gelDThis study
FMME-GG07FMME-GG01 ΔldhA::P tac -gelAThis study
FMME-GG08FMME-GG07 ΔpoxB::P tac -gelKThis study
Plasmids
pBBR1 MCS2pBBR1 oriV, Kanr, P T7, lacZaPurchased from Addgene
pLO3RP4 oriT, ColE1 ori, Tcr, SacBLab stock
pRK2013RP4 oriT, Kanr, oriT, trfALab stock
pJYSpSC101 ori, Fncpf1, P J23119Purchased from Addgene
pSPINpBBR1 oriV, CmR, P J23119Purchased from Addgene
pET28a-eGFPpET28a ori，Kanr，P tacLab stock
pBBR1-101oripSC101 ori, Kanr, P T7, lacZaThis study
pBBR1-pK18oripK18 ori, Kanr, P T7, lacZaThis study
pBBR1-RK2oriRK2 ori, Kanr, P T7, lacZaThis study
pBBR1-ColE1oriColE1 ori, Kanr, P T7, lacZaThis study
pBBR1-pET28aoripET28a ori, Kanr, P T7, lacZaThis study
pBBR1-pVS1oripVS1 ori, Kanr, P T7, lacZaThis study
pCas9pSC101 ori, AmprPurchased from Addgene
pSGN20pET28a ori, Kanr, SacBPurchased from Addgene
pCaspSC101 ori, CmrThis study
pN20pBBR1 oriV, Kanr, SacBThis study
pN20-ldhApBBR1 oriV, Kanr, SacB, N20This study
pRecET-Cre/loxPpBBR1 carrying lox-CmR-lox, CreThis study
pBBR1 MCS2-P tac -eGFPpBBR1 carrying P tac promoterThis study
pBBR1 MCS2-P trc -eGFPpBBR1 carrying P trc promotorThis study
pBBR1 MCS2-P J23119 -eGFPpBBR1 carrying P J23119 promotorThis study
pBBR1 MCS2-P araB -eGFPpBBR1 carrying P araB promotorThis study
pBBR1 MCS2-P 1 -eGFPpBBR1 carrying P 1 promotorThis study
pBBR1 MCS2-P 51 -eGFPpBBR1 carrying P 51 promotorThis study
pLO3-eGFPpLO3, P J23119, eGFPThis study
pCpf1-eGFPpCpf1, P J23119, eGFPThis study
pSPIN-eGFPpSPIN, P J23119, eGFPThis study
pBBR1 MCS2-P tac -pgmpBBR1 carrying P tac -pgm cassetteThis study
pBBR1 MCS2-P tac -vgbpBBR1 carrying P tac -vgb cassetteThis study
pBBR1 MCS2-P tac -gelApBBR1 carrying P tac -gelA cassetteThis study
pBBR1 MCS2-P tac -gelKpBBR1 carrying P tac -gelK cassetteThis study
pBBR1 MCS2-P tac -gelDpBBR1 carrying P tac -gelD cassetteThis study
), ArticleFig(id=1227681737157836956, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=表1, caption=

本研究使用的菌株和质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
NamesRelevant characteristicsSources
Strains
E. coli JM109E. coli JM109 wild-type strainLab stock
E. coli S17-1E. coli S17-1 carrying pLO3Lab stock
E. coli HB101E. coli HB101 carrying pRK2013Lab stock
FMME-GG01Wild-type Sphingomonas paucimobilisLab stock
FMME-GG02FMME-GG01 carrying pBBR1-pgmThis study
FMME-GG03FMME-GG01 carrying pBBR1-vgbThis study
FMME-GG04FMME-GG01 carrying pBBR1-gelAThis study
FMME-GG05FMME-GG01 carrying pBBR1-gelKThis study
FMME-GG06FMME-GG01 carrying pBBR1-gelDThis study
FMME-GG07FMME-GG01 ΔldhA::P tac -gelAThis study
FMME-GG08FMME-GG07 ΔpoxB::P tac -gelKThis study
Plasmids
pBBR1 MCS2pBBR1 oriV, Kanr, P T7, lacZaPurchased from Addgene
pLO3RP4 oriT, ColE1 ori, Tcr, SacBLab stock
pRK2013RP4 oriT, Kanr, oriT, trfALab stock
pJYSpSC101 ori, Fncpf1, P J23119Purchased from Addgene
pSPINpBBR1 oriV, CmR, P J23119Purchased from Addgene
pET28a-eGFPpET28a ori，Kanr，P tacLab stock
pBBR1-101oripSC101 ori, Kanr, P T7, lacZaThis study
pBBR1-pK18oripK18 ori, Kanr, P T7, lacZaThis study
pBBR1-RK2oriRK2 ori, Kanr, P T7, lacZaThis study
pBBR1-ColE1oriColE1 ori, Kanr, P T7, lacZaThis study
pBBR1-pET28aoripET28a ori, Kanr, P T7, lacZaThis study
pBBR1-pVS1oripVS1 ori, Kanr, P T7, lacZaThis study
pCas9pSC101 ori, AmprPurchased from Addgene
pSGN20pET28a ori, Kanr, SacBPurchased from Addgene
pCaspSC101 ori, CmrThis study
pN20pBBR1 oriV, Kanr, SacBThis study
pN20-ldhApBBR1 oriV, Kanr, SacB, N20This study
pRecET-Cre/loxPpBBR1 carrying lox-CmR-lox, CreThis study
pBBR1 MCS2-P tac -eGFPpBBR1 carrying P tac promoterThis study
pBBR1 MCS2-P trc -eGFPpBBR1 carrying P trc promotorThis study
pBBR1 MCS2-P J23119 -eGFPpBBR1 carrying P J23119 promotorThis study
pBBR1 MCS2-P araB -eGFPpBBR1 carrying P araB promotorThis study
pBBR1 MCS2-P 1 -eGFPpBBR1 carrying P 1 promotorThis study
pBBR1 MCS2-P 51 -eGFPpBBR1 carrying P 51 promotorThis study
pLO3-eGFPpLO3, P J23119, eGFPThis study
pCpf1-eGFPpCpf1, P J23119, eGFPThis study
pSPIN-eGFPpSPIN, P J23119, eGFPThis study
pBBR1 MCS2-P tac -pgmpBBR1 carrying P tac -pgm cassetteThis study
pBBR1 MCS2-P tac -vgbpBBR1 carrying P tac -vgb cassetteThis study
pBBR1 MCS2-P tac -gelApBBR1 carrying P tac -gelA cassetteThis study
pBBR1 MCS2-P tac -gelKpBBR1 carrying P tac -gelK cassetteThis study
pBBR1 MCS2-P tac -gelDpBBR1 carrying P tac -gelD cassetteThis study
), ArticleFig(id=1227681737241723043, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=EN, label=Table 2, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimers sequence (5′→3′)
pBBR1-FCTCAAATGCCTGAGGCCAGTTTG
pBBR1-RAAGCACACGGTCACACTGCTTCC
tac-eGFP-FCTGGCCTCAGGCATTTGAGTTGACAATTAATCATCGGCTCG
tac-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
trc-eGFP-FCTGGCCTCAGGCATTTGAGTTGACAATTAATCATCCGGCTCGTA
trc-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
J23119-eGFP-FACTGGCCTCAGGCATTTGAGTTGACAGCTAGCTCAGTCCTAGG
J23119-eGFP-RAGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
araB-eGFP-FACTGGCCTCAGGCATTTGAGAAGAAACCAATTGTCCATATTGCATC
araB-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
P1-eGFP-FCTGGCCTCAGGCATTTGAGTGGCGTGCAAATATCTCTAACGAAC
P1-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
P51-eGFP-FCTGGCCTCAGGCATTTGAGCCGGTAGCAAATGGGGGCCGCTTTGTAG
P51-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
pBBR1-RecET-FGCCTGGGGTGCCTAATGAGTGA
pBBR1-RecET-RGATATCGAATTCCTGCAGCCC
SacB-RecET-FGGCTGCAGGAATTCGATATCCACATATACCTGCCGTTCACT
SacB-RecET-RAGCCGATGATTAATTGTCAATTATTTGTTAACTGTTAATTGTCCTTG
RecET-FTTGACAATTAATCATCGGCTCG
RecET-RTCACTGGCCAATCTCCCGGC
arac-FGCCGGGAGATTGGCCAGTGATTATGACAACTTGACGGCTAC
arac-RACGGTCAGTAAATTGGACATATGGAGAAACAGTAGAGAGTTGC
Cre-RecET-FGCCGGGAGATTGGCCAGTGATTATGACAACTTGACGGCTAC
Cre-RecET-RATCGCCATCTTCCAGCAGGCGCAC
lacI-RecET-FCCGGAAGCATAAAGTGTAAATCACTGCCCGCTTTCCAGTC
lacI-RecET-RACTCATTAGGCACCCCAGGCGACACCATCGAATGGCGCAAAACCT
pBBR1-ori-FAAGGCCGCGTTGCTGGCGTTCTACCGGCGCGGCAGCGTGA
pBBR1-ori-RGAAAAGATCAAAGGATCTTCGCGGCCACCGGCTGGCTCGCTTC
pCas9-FACTACTTTAGTCAGTTCCGCAGTA
pCas9-RGGTTCTTATGGCTCTTGTATCTATC
pSC101-ori-FGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTAGCCAG
pSC101-ori-RGCGGAACTGACTAAAGTAGTGAGTTATACACAGGGCTGGGATC
pSGN20-FGAAGATCCTTTGATCTTTTCTACGG
pSGN20-RAACGCCAGCAACGCGGCCTTTTTAC
pJYS-FGGCCCGGTGAACAGTTGTTCTAC
pJYS-RCTAGATTGACAGCTAGCTCAGTCC
UP-RecET-FGCATCGTCTCGCAGACCTCGAGGCTG
UP-RecET -RACATCCAAGCCCCTCCCCTTCAGGC
Down-RecET-FCAGCGGCTCCAGATCGCGGAAAGGG
Down-RecET-RCGGATCGAAGCCTATGCCGATGCCG
eGFP-RecET-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-RecET-RCGGTTGGGAATGTAATTCAGCTTTACTTGTACAGCTCGTCCATGCC
CmR-RecET-FAGCTGAATTACATTCCCAACCGCG
CmR-RecET-RCTTTCCGCGATCTGGAGCCGCTGCAACTTAAATGTGAAAGTGGGTC
pSPIN-ldhA-FCGAGGTCATTTCCGGGGATCCCATATCGTCAATTATTACCTCCACG
pSPIN-ldhA-FCACCAATAACTGCCTTAAAAAAATTACTTGTACAGCTCGTCCATGCC
pSPIN-FGGCAGTTATTGGTGCCCTTCTAGA
pSPIN-RGGATCCCCGGAAATGACCTCGAGG
eGFP-pSPIN-FCATATCGTCAATTATTACCTCCACG
eGFP-pSPIN-RTTACTTGTACAGCTCGTCCATGCC
UP-cas-FGCATCGTCTCGCAGACCTCGAGGCTG
UP-cas-RACATCCAAGCCCCTCCCCTTCAGGC
Down-cas-FCAGCGGCTCCAGATCGCGGAAAGGG
Down-cas-RCGGATCGAAGCCTATGCCGATGCCG
eGFP-cas-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-cas-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pN20-ldhA-FGGAAAACTGTTCCGCGCCTTGTTTTAGAGCTAGAAATAGCAAG
pN20-ldhA-RAAGGCGCGGAACAGTTTTCCACTAGTATTATACCTAGGACTGAG
pJYS101-ldhA-FAAGGCGCGGAACAGTTTTCCCCGGATCTACAACAGTAGAAATTCGGATCCAT
pJYS101-ldhA-RCCGGGGAAAACTGTTCCGCGCCTTATTTAAATAAAACGAAAGGCTCAGTCG
UP-ldhA-500-FCACCACCGAGTTGATTTCCTGGATGC
UP-ldhA-500-RCGTGAGCCTTCCTTTCGTTGCCGCG
Down-ldhA-500-FTCAGCCCGGCCGCTTCTCGAACGTC
Down-ldhA-500-RGTTCGAGGAGTTCCTCCACGAAGC
eGFP-pJYS-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-pJYS-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pLO3-FTCTAGAGTCGACTGTTTAAACCTGCAG
pLO3-RGAGCTCGAATTAAAGGATCTAGGTGAAG
UP-pLO3-FCACCTAGATCCTTTAATTCGAGCTCAATAGCCGGCGATGTTGAGCAGCCC
UP-pLO3-RACATCCAAGCCCCTCCCCTTCAGG
Down-pLO3-FTCAGCGGCTCCAGATCGCGGAAA
Down-pLO3-RTTTAAACAGTCGACTCTAGAACACCTATTACAGCGACACGC
eGFP-pLO3-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-pLO3-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pBBR1-pgm-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-pgm-RAGGTCGACGGTATCGATAAGGCTTTCGTCGGGATGACAGAAGTAGG
pBBR1-vgb-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-vgb-RGAGGTCGACGGTATCGATAAGCTCGAGTTACTCGACGGCCTG
pBBR1-gelA-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelA-RGAGGTCGACGGTATCGATAAGCGCCCAGCGACAAGCATGGTTAAC
pBBR1-gelK-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelK-RCTCGAGGTCGACGGTATCGATAAGTTGTAGTGCGGGATGACGACGCTG
pBBR1-gelD-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelD-RGAGGTCGACGGTATCGATAAGGTGTGCCTCGTCCGCTGCCATCATTG
pN20-poxB-FCATGCACGCGATCCGCGTCGGTTTTAGAGCTAGAAATAGCAAG
pN20-poxB-RCGACGCGGATCGCGTGCATGACTAGTATTATACCTAGGACTGAG
UP-poxB-FGACCGTAGCAGCGTTGATGACCGAC
UP-poxB-RGAGCCGATGATTAATTGTCAAGCTCAGCCGCTCGGCGAGCTGCTG
Down-poxB-FGTCGTCATCCCGCACTACAATCAGCCGGTGCGGCCTTGCGCCTCTTC
Down-poxB-RGAGCGACGAGCCCTTCCTGCTG
), ArticleFig(id=1227681737350774949, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226554101106913370, language=CN, label=表2, caption=

本研究使用的引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimers sequence (5′→3′)
pBBR1-FCTCAAATGCCTGAGGCCAGTTTG
pBBR1-RAAGCACACGGTCACACTGCTTCC
tac-eGFP-FCTGGCCTCAGGCATTTGAGTTGACAATTAATCATCGGCTCG
tac-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
trc-eGFP-FCTGGCCTCAGGCATTTGAGTTGACAATTAATCATCCGGCTCGTA
trc-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
J23119-eGFP-FACTGGCCTCAGGCATTTGAGTTGACAGCTAGCTCAGTCCTAGG
J23119-eGFP-RAGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
araB-eGFP-FACTGGCCTCAGGCATTTGAGAAGAAACCAATTGTCCATATTGCATC
araB-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
P1-eGFP-FCTGGCCTCAGGCATTTGAGTGGCGTGCAAATATCTCTAACGAAC
P1-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
P51-eGFP-FCTGGCCTCAGGCATTTGAGCCGGTAGCAAATGGGGGCCGCTTTGTAG
P51-eGFP-RGCAGTGTGACCGTGTGCTTTTACTTGTACAGCTCGTCCATGCC
pBBR1-RecET-FGCCTGGGGTGCCTAATGAGTGA
pBBR1-RecET-RGATATCGAATTCCTGCAGCCC
SacB-RecET-FGGCTGCAGGAATTCGATATCCACATATACCTGCCGTTCACT
SacB-RecET-RAGCCGATGATTAATTGTCAATTATTTGTTAACTGTTAATTGTCCTTG
RecET-FTTGACAATTAATCATCGGCTCG
RecET-RTCACTGGCCAATCTCCCGGC
arac-FGCCGGGAGATTGGCCAGTGATTATGACAACTTGACGGCTAC
arac-RACGGTCAGTAAATTGGACATATGGAGAAACAGTAGAGAGTTGC
Cre-RecET-FGCCGGGAGATTGGCCAGTGATTATGACAACTTGACGGCTAC
Cre-RecET-RATCGCCATCTTCCAGCAGGCGCAC
lacI-RecET-FCCGGAAGCATAAAGTGTAAATCACTGCCCGCTTTCCAGTC
lacI-RecET-RACTCATTAGGCACCCCAGGCGACACCATCGAATGGCGCAAAACCT
pBBR1-ori-FAAGGCCGCGTTGCTGGCGTTCTACCGGCGCGGCAGCGTGA
pBBR1-ori-RGAAAAGATCAAAGGATCTTCGCGGCCACCGGCTGGCTCGCTTC
pCas9-FACTACTTTAGTCAGTTCCGCAGTA
pCas9-RGGTTCTTATGGCTCTTGTATCTATC
pSC101-ori-FGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTAGCCAG
pSC101-ori-RGCGGAACTGACTAAAGTAGTGAGTTATACACAGGGCTGGGATC
pSGN20-FGAAGATCCTTTGATCTTTTCTACGG
pSGN20-RAACGCCAGCAACGCGGCCTTTTTAC
pJYS-FGGCCCGGTGAACAGTTGTTCTAC
pJYS-RCTAGATTGACAGCTAGCTCAGTCC
UP-RecET-FGCATCGTCTCGCAGACCTCGAGGCTG
UP-RecET -RACATCCAAGCCCCTCCCCTTCAGGC
Down-RecET-FCAGCGGCTCCAGATCGCGGAAAGGG
Down-RecET-RCGGATCGAAGCCTATGCCGATGCCG
eGFP-RecET-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-RecET-RCGGTTGGGAATGTAATTCAGCTTTACTTGTACAGCTCGTCCATGCC
CmR-RecET-FAGCTGAATTACATTCCCAACCGCG
CmR-RecET-RCTTTCCGCGATCTGGAGCCGCTGCAACTTAAATGTGAAAGTGGGTC
pSPIN-ldhA-FCGAGGTCATTTCCGGGGATCCCATATCGTCAATTATTACCTCCACG
pSPIN-ldhA-FCACCAATAACTGCCTTAAAAAAATTACTTGTACAGCTCGTCCATGCC
pSPIN-FGGCAGTTATTGGTGCCCTTCTAGA
pSPIN-RGGATCCCCGGAAATGACCTCGAGG
eGFP-pSPIN-FCATATCGTCAATTATTACCTCCACG
eGFP-pSPIN-RTTACTTGTACAGCTCGTCCATGCC
UP-cas-FGCATCGTCTCGCAGACCTCGAGGCTG
UP-cas-RACATCCAAGCCCCTCCCCTTCAGGC
Down-cas-FCAGCGGCTCCAGATCGCGGAAAGGG
Down-cas-RCGGATCGAAGCCTATGCCGATGCCG
eGFP-cas-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-cas-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pN20-ldhA-FGGAAAACTGTTCCGCGCCTTGTTTTAGAGCTAGAAATAGCAAG
pN20-ldhA-RAAGGCGCGGAACAGTTTTCCACTAGTATTATACCTAGGACTGAG
pJYS101-ldhA-FAAGGCGCGGAACAGTTTTCCCCGGATCTACAACAGTAGAAATTCGGATCCAT
pJYS101-ldhA-RCCGGGGAAAACTGTTCCGCGCCTTATTTAAATAAAACGAAAGGCTCAGTCG
UP-ldhA-500-FCACCACCGAGTTGATTTCCTGGATGC
UP-ldhA-500-RCGTGAGCCTTCCTTTCGTTGCCGCG
Down-ldhA-500-FTCAGCCCGGCCGCTTCTCGAACGTC
Down-ldhA-500-RGTTCGAGGAGTTCCTCCACGAAGC
eGFP-pJYS-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-pJYS-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pLO3-FTCTAGAGTCGACTGTTTAAACCTGCAG
pLO3-RGAGCTCGAATTAAAGGATCTAGGTGAAG
UP-pLO3-FCACCTAGATCCTTTAATTCGAGCTCAATAGCCGGCGATGTTGAGCAGCCC
UP-pLO3-RACATCCAAGCCCCTCCCCTTCAGG
Down-pLO3-FTCAGCGGCTCCAGATCGCGGAAA
Down-pLO3-RTTTAAACAGTCGACTCTAGAACACCTATTACAGCGACACGC
eGFP-pLO3-FGAAGGGGAGGGGCTTGGATGTCATATCGTCAATTATTACCTCCACG
eGFP-pLO3-RCTTTCCGCGATCTGGAGCCGCTGTTACTTGTACAGCTCGTCCATGCC
pBBR1-pgm-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-pgm-RAGGTCGACGGTATCGATAAGGCTTTCGTCGGGATGACAGAAGTAGG
pBBR1-vgb-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-vgb-RGAGGTCGACGGTATCGATAAGCTCGAGTTACTCGACGGCCTG
pBBR1-gelA-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelA-RGAGGTCGACGGTATCGATAAGCGCCCAGCGACAAGCATGGTTAAC
pBBR1-gelK-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelK-RCTCGAGGTCGACGGTATCGATAAGTTGTAGTGCGGGATGACGACGCTG
pBBR1-gelD-FCTGCAGGAATTCGATATCAAGTTGACAATTAATCATCGGCTCG
pBBR1-gelD-RGAGGTCGACGGTATCGATAAGGTGTGCCTCGTCCGCTGCCATCATTG
pN20-poxB-FCATGCACGCGATCCGCGTCGGTTTTAGAGCTAGAAATAGCAAG
pN20-poxB-RCGACGCGGATCGCGTGCATGACTAGTATTATACCTAGGACTGAG
UP-poxB-FGACCGTAGCAGCGTTGATGACCGAC
UP-poxB-RGAGCCGATGATTAATTGTCAAGCTCAGCCGCTCGGCGAGCTGCTG
Down-poxB-FGTCGTCATCCCGCACTACAATCAGCCGGTGCGGCCTTGCGCCTCTTC
Down-poxB-RGAGCGACGAGCCCTTCCTGCTG
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代谢工程改造少动鞘氨醇单胞菌发酵生产结冷胶
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孙鹏 1 , 薛正莲 1 , 高聪 2 , 刘佳 2 , 吴静 2 , 李晓敏 2 , 刘立明 2
微生物学报 | 研究报告 2025,65(7): 3056-3074
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微生物学报 | 研究报告 2025, 65(7): 3056-3074
代谢工程改造少动鞘氨醇单胞菌发酵生产结冷胶
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孙鹏1, 薛正莲1, 高聪2, 刘佳2, 吴静2, 李晓敏2 , 刘立明2
作者信息
  • 1.安徽工程大学 生物与食品工程学院，安徽省工业微生物分子育种工程实验室，安徽 芜湖
  • 2.江南大学 生物工程学院，工业生物技术教育部重点实验室，江苏 无锡
Metabolic engineering of Sphingomonas paucimobilis for the production of gellan gum
Peng SUN1, Zhenglian XUE1, Cong GAO2, Jia LIU2, Jing WU2, Xiaomin LI2 , Liming LIU2
Affiliations
  • 1.Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, School of Biology and Food Engineering, Anhui Polytechnic University, Wuhu, Anhui, China
  • 2.Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
出版时间: 2025-07-04 doi: 10.13343/j.cnki.wsxb.20240844
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摘要
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【目的】 以少动鞘氨醇单胞菌为出发菌株，通过代谢工程改造构建高产结冷胶工程菌株，并优化发酵工艺，为利用该菌高效合成结冷胶提供理论支撑与工艺基础。 【方法】 基于CRISPR-Cas9系统开发适用于少动鞘氨醇单胞菌的基因编辑工具，并利用该工具在基因组中整合组成型启动子驱动的结冷胶合成基因簇调控蛋白基因(gelA)和β-1,4-葡萄糖醛酸转移酶基因(gelK)。在此基础上，通过单因素试验优化了碳源、氮源、pH及溶氧等发酵工艺参数。 【结果】 工程菌株FMME-GG08在摇瓶培养中结冷胶产量达到10.8 g/L，较出发菌株提升130.2%。发酵工艺优化后，在15 L规模发酵罐中结冷胶产量达到20.1 g/L，蔗糖转化效率为0.50 g/g。 【结论】 本研究不仅成功构建了结冷胶高产菌株并建立了高效发酵工艺，为工业化生产提供了可靠技术方案，同时所开发的基因编辑策略也为代谢工程改造少动鞘氨醇单胞菌等非模式微生物生产其他高价值胞外多糖提供了重要参考。

结冷胶  /  少动鞘氨醇单胞菌  /  代谢工程  /  CRISPR-Cas9

[Objective] Using Sphingomonas paucimobilis as the starting strain, a high-yield gellan gum-producing engineered strain was constructed through metabolic engineering, and fermentation process optimization was performed, providing both theoretical support and technical foundations for the efficient biosynthesis of gellan gum with this bacterium. [Methods] A CRISPR-Cas9-based gene editing system was developed for S. paucimobilis, and subsequently employed to genomically integrate two key gellan gum biosynthesis genes: the regulatory protein gene (gelA) and the β-1,4-glucuronosyltransferase gene (gelK), both under the control of constitutive promoters. Building upon this foundation, fermentation parameters including carbon source, nitrogen source, pH, and dissolved oxygen were systematically optimized through single-factor experiments. [Results] The engineered strain FMME-GG08 achieved a gellan gum yield of 10.8 g/L in shake-flask cultivation, representing a 130.2% enhancement over the parental strain. Following fermentation process optimization, the production level reached 20.1 g/L in 15 L scale bioreactors, with a sucrose conversion efficiency of 0.50 g/g. [Conclusion] This study not only successfully constructed a high-yield gellan gum-producing strain and established an efficient fermentation process, providing a reliable technical solution for industrial production, but also developed a genetic editing strategy that serves as an important reference for metabolic engineering of non-model microorganisms to produce other high-value exopolysaccharides.

gellan gum  /  Sphingomonas paucimobilis  /  metabolic engineering  /  CRISPR-Cas9
孙鹏, 薛正莲, 高聪, 刘佳, 吴静, 李晓敏, 刘立明. 代谢工程改造少动鞘氨醇单胞菌发酵生产结冷胶. 微生物学报, 2025 , 65 (7) : 3056 -3074 . DOI: 10.13343/j.cnki.wsxb.20240844
Peng SUN, Zhenglian XUE, Cong GAO, Jia LIU, Jing WU, Xiaomin LI, Liming LIU. Metabolic engineering of Sphingomonas paucimobilis for the production of gellan gum[J]. Acta Microbiologica Sinica, 2025 , 65 (7) : 3056 -3074 . DOI: 10.13343/j.cnki.wsxb.20240844
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结冷胶(gellan gum)是一种由d-葡萄糖、d-葡萄糖醛酸和l-鼠李糖按2:1:1连接构成的线性四糖重复单元聚合体。结冷胶的葡萄糖残基通常由乙酰基和甘油酰基修饰(图1)，平均相对分子质量约为500 kDa[1]。结冷胶具有良好的流变性和凝胶特性[2]，且易于降解、安全无毒[3]，可作为增稠剂、凝结剂、悬浮剂和成膜剂等广泛应用于食品、医药和化妆品等领域[4-6]。
目前，结冷胶主要通过少动鞘氨醇单胞菌(Sphingomonas paucimobilis)发酵生产，少动鞘氨醇单胞菌生产菌株中的结冷胶合成途径主要包括3个模块：糖核苷酸前体的合成、四糖单元的组装、四糖单元的聚合与输出(图1)，其途径酶的编码基因成簇排列在基因组上[7]。然而，目前通过少动鞘氨醇单胞菌发酵法合成结冷胶的生产效率较低，限制了其进一步规模化应用[8]。为提高生产效率以满足市场对结冷胶的需求，研究人员已通过代谢工程改造结冷胶高产菌株或优化发酵条件等方式提高结冷胶产量。构建高产菌株的代谢工程方法主要有：强化结冷胶合成路径相关基因[9]，敲除旁支途径相关基因[10]，强化结冷胶转运系统[11]，异源表达透明颤菌血红蛋白[12]等。此外，还可结合物理、化学诱变和细胞融合等传统方法[13]进一步提升结冷胶产量。
少动鞘氨醇单胞菌作为非模式菌株，相较于大肠杆菌、谷氨酸棒状杆菌、枯草芽孢杆菌等工业菌株缺乏成熟的基因编辑工具，目前仅有少数研究能通过三亲本接合对少动鞘氨醇单胞菌进行基因编辑[14-15]。然而，该方法基因编辑效率低，筛选验证过程复杂，且须多次传代才能实现基因的稳定表达[16-18]，仍存在很大的局限性，阻碍了结冷胶生产菌种的快速更新迭代。
随着基因编辑技术的发展，微生物遗传改造的效率和精度大大超越了传统方法，每种基因编辑技术具有不同的特点。RecET-Cre/loxP系统在多基因编辑和复杂基因组改造中表现优越，适用于更复杂的遗传工程，但其操作步骤相对繁琐且需要精细地设计[19]；以CRISPR-Cas9和CRISPR-Cpf1为代表的CRISPR-Cas编辑工具同时兼具高效性与特异性，能够在短时间内实现长链DNA的敲除与插入，且操作相对简便，但存在潜在脱靶效应[20-22]。引导RNA辅助靶向的转座元件插入(INsert transposable elements by guide RNA-assisted TargEting, INTEGRATE)技术在特定插入和稳定表达方面表现出色，但其应用范围受限于特定宿主和转座子的选择[23]。然而，上述基因编辑方法仍未应用于少动鞘氨醇单胞菌，这对代谢工程改造少动鞘氨醇单胞菌高效合成结冷胶产生了不利影响，因此针对少动鞘氨醇单胞菌开发高效的基因工程操作工具变得尤为重要。
本研究首先在少动鞘氨醇单胞菌中筛选了以CRISPR-Cas为代表的新一代基因编辑系统，并基于宿主特点对基因编辑系统进行特异性改造，最终建立了适用于少动鞘氨醇单胞菌的基因编辑方法。利用该方法对本实验室保藏的一株产结冷胶的少动鞘氨醇单胞菌FMME-GG01进行了代谢工程改造，即对结冷胶合成路径的限速酶编码基因——结冷胶合成基因簇调控蛋白基因(gelA)、β-1,4-葡萄糖醛酸转移酶基因(gelK)进行过表达。经过工艺优化后，在15 L发酵罐中进行分批补料发酵，以期为结冷胶的工业化生产奠定基础。
1 材料与方法
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1.1 材料
1.1.1 菌株和质粒
本研究中使用的菌株和质粒如表1所示。
1.1.2 培养基
LB培养基(g/L)：蛋白胨10.0，酵母粉5.0，氯化钠10.0。固体培养基中添加琼脂粉20.0 g/L。
YPG培养基(g/L)：葡萄糖20.0，酵母膏3.0，蛋白胨5.0，琼脂粉20.0。
YM固体培养基(g/L)：葡萄糖10.0，蛋白胨5.0，麦芽抽提物3.0，酵母粉3.0，琼脂粉20.0。
种子斜面培养基(g/L)：蔗糖10.0，蛋白胨5.0，酵母膏3.0，牛肉膏2.0，氯化钠5.0，琼脂粉20.0。
种子培养基(g/L)：蔗糖20.0，蛋白胨5.0，酵母粉3.0，氯化钠5.0。
发酵培养基(g/L)：蔗糖40.0，豆粕粉3.0，氯化铵1.0，磷酸氢二钾2.0，七水硫酸镁1.2，轻质碳酸钙1.0。
以上培养基均使用氢氧化钠调pH至7.0-7.2，蔗糖与葡萄糖的灭菌条件为115 ℃、15 min，其余组分均在121 ℃条件下灭菌20 min。
1.1.3 主要试剂和仪器
葡萄糖、蔗糖、氯化钠、琼脂粉、磷酸氢二钾、七水硫酸镁等试剂均购自国药集团化学试剂上海有限公司；蛋白胨、酵母粉等试剂均购自Oxoid公司；豆粕粉购自山东万得福生物科技有限公司。
Gene Pulser Xcell电穿孔仪，伯乐生命医学产品(上海)有限公司；往复式振荡培养箱，太仓市强文实验设备有限公司；NanoDrop分光光度计，赛默飞世尔科技(中国)有限公司；紫外可见分光光度计，岛津仪器(苏州)有限公司；黏度计，阿美特克-博勒飞公司；15 L不锈钢发酵罐，上海百仑生物科技有限公司。
1.1.4 引物
本研究所用引物见表2，由苏州金唯智生物科技有限公司合成。
1.2 基因编辑方法的建立
1.2.1 少动鞘氨醇单胞菌转化
从甘油保藏管中吸取20 μL菌液，稀释后涂布在YM平板上，30 ℃恒温培养至平板上长满菌落。使用无菌水洗脱平板上的菌体，冰浴15 min后，4 ℃、6 000 r/min离心5 min，收集菌体。用预冷的10%甘油洗涤3次后，加入200 μL预冷的10%甘油，重悬菌体，分装成每管100 μL的感受态细胞，-80 ℃保存备用。
向感受态细胞中加入2 μg待转化质粒，混匀后加入电击杯中，置于冰上预冷10 min，将电穿孔仪电压分别设置为1 600、1 800、2 000、2 200、2 500 V/mm 这5个梯度。电击结束后，向电击杯中加入900 μL YPG培养基，转移至无菌EP管中，30 ℃、220 r/min培养1 h后，6 000 r/min离心2 min，弃上清，重悬后涂布于含有50 mg/L卡那霉素的YM固体培养基，30 ℃恒温培养48 h。
1.2.2 复制子筛选
六个不同来源的复制子pSC101ori、pK18ori、RK2ori、ColE1ori、pET28aori和pVS1ori由苏州金唯智生物科技有限公司合成。使用引物对pBBR1-F/R以质粒pBBR1 MCS2为模板，PCR获得线性化质粒片段。PCR反应体系(50 μL)：2×Phanta Flash Master Mix (Dye Plus)(p520) 25 µL，上、下游引物(10 µmol/L)各4 µL，DNA模板2 µL，ddH2O 15 µL。PCR反应条件：98 °C预变性1 min；98 °C变性10 s，55 °C退火15 s，72 °C延伸1 min，32个循环；72 °C终延伸5 min。将该片段与不同复制子片段经同源重组连接后转化E. coli JM109，获得重组质粒pBBR1-101ori、pBBR1-pK18ori、pBBR1-RK2ori、pBBR1-ColE1ori、pBBR1-pET28aori和pBBR1-pVS1ori。将重组质粒分别转化FMME-GG01感受态，涂布在含有50 mg/L卡那霉素的YM固体培养基上，根据转化子数量，确定最佳复制子。
1.2.3 启动子筛选
根据本实验室前期研究经验，选取6个启动子P tac 、P trc 、P J23119 、P araB 、P 1 和P 51，删除其中的诱导调控序列，由苏州金唯智生物科技有限公司合成。分别使用引物对tac-eGFP-F/R、trc-eGFP-F/R、J23119-eGFP-F/R、araB-eGFP-F/R、P1-eGFP-F/R、P51-eGFP-F/R，以不同启动子基因和eGFP为模板进行融合PCR。PCR反应体系(50 μL)：2×Phanta Flash Master Mix (Dye Plus) (p520) 25 µL，上、下游引物(10 µmol/L)各2 µL，启动子模板4 µL，eGFP模板2 µL，ddH2O 15 µL。PCR反应条件：98 °C预变性1 min；98 °C变性10 s，55 °C退火15 s，72 °C延伸1 min，32个循环；72 °C终延伸5 min。构建不同启动子驱动的eGFP表达框。使用引物对pBBR1-F/R以质粒pBBR1 MCS2为模板，PCR获得带有eGFP表达框同源臂的线性化质粒片段，将该片段与各eGFP表达框经同源重组连接后转化至E. coli JM109，获得重组质粒pBBR1 MCS2-P tac -eGFP、pBBR1 MCS2-P trc -eGFP、pBBR1 MCS2-P araB -eGFP、pBBR1 MCS2-P J23119 -eGFP、pBBR1 MCS2-P 1 -eGFP、pBBR1 MCS2-P 51 -eGFP，并分别转化至FMME-GG01感受态，涂布在含有50 mg/L卡那霉素的YM固体培养基上，挑取单菌落接种至YPG液体培养基，根据菌液荧光强度/OD600比值确定启动子强度。
1.2.4 基因编辑系统的构建与评估
分别使用pSC101和pBBR1 MCS2质粒复制子，替换INTEGRATE、CRISPR-Cpf1、RecET- Cre/loxP、CRISPR-Cas9 这4种基因编辑系统质粒原有的复制子序列，构建可以在少动鞘氨醇单胞菌中使用的基因编辑系统。选用ldhA基因作为中性位点，利用P tac 启动子驱动的eGFP荧光蛋白基因整合框，评估各编辑系统的编辑效率。
1.3 重组菌株的构建
1.3.1 关键基因筛选
根据先前研究，选择5个参与结冷胶合成的基因，以少动鞘氨醇单胞菌结冷胶基因组为模板，分别使用对应的引物对扩增，经同源重组克隆至pBBR1 MCS2质粒，获得重组表达质粒pBBR1 MCS2-P tac -pgm、pBBR1 MCS2-P tac -vgb、pBBR1 MCS2-P tac -gelA、pBBR1 MCS2-P tac -gelK和pBBR1 MCS2-P tac -gelD，并分别转化至FMME-GG01感受态，构建重组菌株FMME-GG02-FMME-GG06。
1.3.2 基因组编辑
利用改造的CRISPR-Cas9系统进行基因组编辑，分别选取ldhA和poxB作为中性位点，将gelA和gelK基因整合至FMME-GG01基因组中。以gelA基因为例，使用引物对UP-ldhA-500-F/R和Down-ldhA-500-F/R分别扩增ldhA基因上下游各500 bp序列；使用引物对UP-ldhA-500-F/Down-ldhA-500-R进行融合PCR构建gelA整合框。将整合框片段与质粒pN20-ldhA按3:1比例混合，转化至含有pCas质粒的FMME-GG01感受态，涂布于含有33 mg/L氯霉素和50 mg/L卡那霉素的YM固体培养基，30 ℃培养48 h，挑选单菌落进行菌落PCR验证。验证正确的转化子进一步通过在培养基中添加15%的蔗糖去除pN20-ldhA质粒，42 ℃培养24 h去除pCas质粒，获得重组菌株FMME-GG07。利用同样的方法构建重组菌株FMME-GG08。
1.4 重组菌株发酵
1.4.1 种子培养
从FMME-GG01甘油中保藏划线于种子斜面，30 ℃培养24 h。使用10 mL无菌水洗斜面，取1 mL转接至种子培养基，30 ℃、140 r/min培养12-16 h。再取1 mL种子液转接至新的种子培养基，30 ℃、140 r/min培养6-8 h。
1.4.2 摇瓶发酵
将10 mL种子液转接至装有90 mL发酵培养基的500 mL带挡板摇瓶中，28 ℃、150 r/min往复式振荡摇床培养60 h。在发酵18-20 h时补加2 mL 500 g/L蔗糖。
1.4.3 15 L罐补料分批发酵
15 L发酵罐初始装液量为9.45 L，将1.05 L种子液接种到发酵罐中。发酵初始条件控制为：温度28 ℃，转速300 r/min，pH 7.5，通气量
1 vvm。发酵罐中的溶氧通过搅拌转速 (300-1 000 r/min)自动维持在30%以上。在发酵18-20 h时补加10 g/L蔗糖。整个发酵过程中通过自动添加质量浓度为30%的NaOH将培养基pH维持在7.0。
1.4.4 相关参数测定
取10 mL发酵液，用去离子水稀释5倍，沸水浴10 min，23 ℃、8 000 r/min离心10 min收集上清。加入3倍体积无水乙醇，4 ℃过夜沉淀，离心收集上清用于残糖测定，收集沉淀置于预烘干并称重的锡箔纸上，在65 ℃烘箱中烘干至恒重，计算结冷胶产量[24]。
将去除结冷胶的发酵液利用冷冻干燥仪冻干，加入等体积去离子水将样品重新溶解，采用苯酚硫酸法测定残糖[25]。
取35 mL发酵液置于50 mL离心管中，使用黏度计测定黏度，测试条件为4号转子、25 ℃、10 r/min[26]。
以内参基因gyrB为参照，利用实时荧光定量PCR检测合成途径中关键基因的表达情况[27]。
2 结果与分析
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2.1 少动鞘氨醇单胞菌基因编辑方法的建立
由于少动鞘氨醇单胞菌产生大量结冷胶包裹在菌体表面，在制备感受态细胞过程中，使用离心法无法有效去除菌体表面的多糖，显著影响了感受态细胞的转化效率。为解决这一问题，本研究从平板中使用无菌水将菌体洗脱下来，有效分离了菌体与其分泌的结冷胶多糖。此外，电击法被证实为最有效的转化方法[28]，本研究使用梯度电压进行电击试验，结果显示(图2)，当电击电压为2 500 V/mm时，转化子数量最多。
目前，除pBBR1 MCS2质粒外，尚无可用于少动鞘氨醇单胞菌的质粒元件。根据调研，选择了6个不同来源的复制子，其中pSC101 ori、pET28a ori、ColE1 ori 和pK18 ori均为大肠杆菌和谷氨酸棒状杆菌中常用的质粒复制子[29-32]，RK2 ori和pVS1 ori为假单胞菌中常用的质粒复制子[33-34]。将不同的复制子分别克隆至pBBR1，替代其原有复制子，并转化至少动鞘氨醇单胞菌，通过抗性筛选可在少动鞘氨醇单胞菌中起始质粒复制的复制子[35]。转化子数量结果如图3A所示，质粒pSC101和pET28a的复制子均能在少动鞘氨醇单胞菌中成功控制质粒复制。相较于pET28a，使用pSC101复制子转化后平板菌落数提升了200%。在FMME-GG01菌株中对不同质粒的拷贝数进行了定量分析(图3B)。结果显示，pET28a质粒的拷贝数为22个/细胞，pSC101质粒的拷贝数为4个/细胞。进一步分析表明，质粒的转化效率与拷贝数之间存在负相关性，即质粒拷贝数越高，其转化效率越低。因此，后续研究使用pSC101复制子改造基因编辑系统。
为了筛选组成型启动子用于代谢工程改造，根据调研，选择了6个启动子，在合成时删除了诱导启动子的诱导序列，以避免整合基因后诱导剂的添加。荧光检测结果显示，P tac 、P trc 、P J23119 、P araB 、P 1 和P 51 启动子都能在少动鞘氨醇单胞菌中启动eGFP表达，其中P tac 、P trc 和P J23119 展现出较强的启动能力，P tac 表现出最高的表达强度(图4)[36]。因此，后续研究中使用P tac 启动子用于重组酶的表达。
将INTEGRATE、CRISPR-Cpf1、RecET-Cre/loxP、CRISPR-Cas9这4种基因编辑系统质粒原有的复制子替换为pSC101和pBBR1 MCS2质粒复制子，使各质粒可在少动鞘氨醇单胞菌中复制表达。将eGFP荧光蛋白基因整合框整合至ldhA位点，评估各编辑系统的编辑效率(图5)。
以三亲本接合系统为对照，评估本研究改造的基因编辑系统。结果表明，CRISPR-Cpf1平板上无荧光菌落；RecET-Cre/loxP平板上有少量荧光菌落(1个)；INTEGRATE与CRISPR-Cas9平板上有较多的荧光菌落，分别为12个和22个(图6A)。相较于INTEGRATE，CRISPR-Cas9在少动鞘氨醇单胞菌中具有更高的编辑效率，从平板上挑选部分单菌落进行聚合酶链式反应(PCR)验证，并进一步实施测序验证(图6B、6C)。在CRISPR-Cas9平板上挑选的13个单菌落中，发现1个菌落的测序结果显示存在单核苷酸位点突变，基因编辑整合效率为92.3%；在INTEGRATE平板上挑选的8个单菌落中，有2个菌株出现核苷酸位点突变，基因编辑整合效率为75.0%。因此，后续研究使用改造的CRISPR-Cas9系统对少动鞘氨醇单胞菌进行基因编辑和代谢工程改造。
2.2 影响结冷胶合成的关键基因确定
为快速筛选能够促进结冷胶积累的关键基因，根据文献调研，选取了1个调控基因，即来源于透明颤菌的促进菌体获取氧气的vgb基因[37-38]，以及少动鞘氨醇单胞菌结冷胶合成路径中的4个内源基因，分别为促进前体物质合成的pgm基因、调控结冷胶合成的gelA基因、促进结冷胶单糖元件组装的gelK基因以及促进结冷胶外排的gelD基因。以pBBR1 MCS2为质粒骨架，构建了各基因的重组表达质粒pBBR1 MCS2-P tac -pgm、pBBR1 MCS2-P tac -vgb、pBBR1 MCS2-P tac -gelA、pBBR1 MCS2-P tac -gelK和pBBR1 MCS2-P tac -gelD，并通过优化的电击法转化至出发菌株FMME-GG01中，最后通过重组菌株的摇瓶发酵评估这些基因对结冷胶积累和发酵液黏度的影响。
摇瓶结果显示(图7)，过表达gelA基因时，结冷胶产量达到7.2 g/L，发酵液黏度为3 780 mPa·s，较对照菌株分别提高了53.1%和49.4%；过表达gelK基因时，结冷胶产量无显著提升，但发酵液黏度达到4 750 mPa·s，较对照菌株提高了87.7%；过表达pgm、vgb和gelD基因对结冷胶的积累和发酵液黏度均无显著影响。上述结果表明gelA和gelK基因对结冷胶合成有重要影响。因此，后续研究将通过基因组编辑，在出发菌株FMME-GG01中增加这2个基因的拷贝数，以构建结冷胶高产菌株。
2.3 结冷胶合成路径强化
基于上述结果，利用本研究改造的CRISPR Cas9系统将gelA整合至FMME-GG01基因组ldhA基因位点，得到重组菌株FMME-GG07。进一步将gelK整合至FMME-GG07基因组poxB基因位点，得到重组菌株FMME-GG08。利用FMME-GG08进行摇瓶发酵，结果如图8所示，在发酵过程中，重组菌株FMME-GG08中gelA和gelK的转录水平均高于出发菌株FMME-GG01，其生长未受到影响；发酵结束后，结冷胶产量达到10.8 g/L，发酵液黏度为4 683 mPa·s，较FMME-GG01分别提高了130.2%和85.1%。
2.4 优化发酵过程参数强化结冷胶合成
为进一步提高结冷胶的产量，在15 L发酵罐中进行放大培养，优化种子培养、碳源、氮源、pH和溶解氧浓度等关键参数。
种子液生长曲线如图9所示，在一级种子摇瓶中，0-10 h为延滞期，10-16 h为对数生长期；16-20 h为稳定期。在二级种子摇瓶中， 0-4 h为延滞期，4-8 h为对数生长期；8-13 h为稳定期。处于对数生长期的菌种对新环境的适应期短，接种后可迅速生长，因此选择培养 6-8 h的二级种子液接种至发酵罐发酵。
分别选取葡萄糖、蔗糖和麦芽糖浆作为唯一碳源，结果如图10A所示，蔗糖作为唯一碳源时，结冷胶产量最高达13.5 g/L。在补糖策略上，设计了2个条件：(1) 初糖浓度40 g/L，发酵过程中不再补加碳源；(2) 初糖浓度30 g/L，发酵20 h时补加蔗糖10 g/L。结果如图10B所示，条件(2)最优，结冷胶产量、产率、生产强度分别为15.1 g/L、0.37 g/g、0.25 g/(L·h)。
基于上述结果，分别选取蛋白胨、豆粕粉、玉米浆和麸皮提取物作为唯一氮源，结果如图10C所示，选取豆粕粉为唯一氮源时，结冷胶产量最高达14.7 g/L，相较于最优的蛋白胨作为氮源产量仅降低了2.6%，但豆粕粉价格更低廉，利于节约工业化生产成本，因此后续研究使用豆粕粉为氮源。进一步，在发酵培养基中使用梯度质量浓度(1‰、2‰、3‰、4‰、5‰、6‰、7‰)豆粕粉，结果如图10D所示，添加质量浓度3‰的豆粕粉更有利于结冷胶积累，此时结冷胶产量、产率、生产强度分别为16.4 g/L、0.41 g/g、0.27 g/(L·h)。
同时，设计了2个pH控制条件：(1) 控制初始pH为7.5，发酵全程不做调控；(2) 初始pH为7.0，发酵过程中使用质量浓度30% NaOH将发酵液pH维持在7.0。结果如图10E所示，条件(2)更利于结冷胶积累，此时结冷胶产量、产率和生产强度分别为17.5 g/L、0.43 g/g、0.29 g/(L·h)。
此外，设计了3个条件优化溶氧，即分别在0-6 h、0-12 h、0-24 h控制转速不高于450 r/min。如图10F所示，在发酵前12 h控制转速不高于450 r/min，后续将转速关联溶氧控制在30%以上时更利于结冷胶积累，此时结冷胶产量、产率和生产强度分别为18.0 g/L、0.45 g/g、0.30 g/(L·h)。
综合上述最优发酵工艺，重组菌株FMME-GG08在15 L发酵罐中发酵60 h，结冷胶产量达到20.1 g/L，是其在摇瓶水平产量的2倍，底物蔗糖的转化率为0.50 g/g，生产强度为0.33 g/(L·h)，发酵液黏度为8 175 mPa·s。
3 讨论与结论
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结冷胶是近年来在食品和医药领域中应用最广泛的凝胶剂之一，1992年美国食品药品监督管理局批准结冷胶可作为凝胶剂、悬浮剂和稳定剂应用于食品和化妆品工业，1996年我国批准结冷胶可作为食品添加剂。目前，微生物发酵法是结冷胶生产中最常见的方法。1982年首次发现少动鞘氨醇单胞菌以来，该菌已广泛应用于结冷胶的工业化发酵生产[8]。参与结冷胶生物合成的基因有26个，但结冷胶生产菌株的代谢调控仅限于少数基因的过表达或敲除。这主要是由于对结冷胶生物合成途径的认识不够清晰，尤其是对四糖重复单元组装机制的研究较少，且缺少高效的遗传操作工具和诱导调控元件。传统的三亲接合效率低、耗时长、目标菌株筛选困难，无法满足菌株构建的需求[39]。因此，本研究基于CRISPR技术构建了高效适用于少动鞘氨醇单胞菌的基因编辑工具，并利用该工具对少动鞘氨醇单胞菌进行代谢工程改造，以提高结冷胶产量，为以非模式微生物为底盘菌株构建高产结冷胶等胞外多糖产品的生产菌株提供了借鉴。
本研究从6种大肠杆菌、谷氨酸棒状杆菌和假单胞菌中常用的质粒复制子元件中筛选能够在少动鞘氨醇单胞菌中复制与表达的复制子。已报道的研究中，仅宽范围宿主质粒pBBR1可在少动鞘氨醇单胞菌中复制与表达。本研究表明，来源于质粒pSC101和pET28a的复制子均可在少动鞘氨醇单胞菌中复制和表达，为构建适用于该菌种的载体质粒提供了新的工具。此外，本研究利用绿色荧光蛋白表征不同启动子的调控强度，筛选出了适用于该菌株的不同强度启动子元件P tac 、P trc 和P J23119，为该菌种的代谢工程改造提供了诱导调控元件。随后，利用复制子元件改造并筛选了5种基因编辑系统，其中INTEGRATE系统和CRISPR-Cas9系统均能有效编辑少动鞘氨醇单胞菌，且CRISPR-Cas9系统表现出更高的编辑效率。利用改造的CRISPR-Cas9系统，在出发菌株基因组上强化合成路径基因gelA和gelK，同时敲除副产物基因ldhA和poxB，构建了FMME-GG08菌株，其摇瓶产量为10.8 g/L。进一步优化碳源、氮源、pH、溶氧等发酵参数，并在15 L发酵罐上进行了放大试验，最终结冷胶的产量达到20.1 g/L，已初步具备工业化生产潜力，但相较于目前报道的最高产量仍存在一定的差距。如何进一步筛选关键基因并通过代谢改造生产菌株以提高结冷胶产量是结冷胶生产待解决的关键问题。
在发酵生产结冷胶过程中，随着产物的积累发酵液的黏度逐渐增加，溶氧水平随之下降，从而抑制了结冷胶的进一步积累。为进一步提高结冷胶的产率，在后续的研究中可以通过外源补加携氧剂H2O2或适量供给无菌氧气来改善发酵过程中的溶氧条件，满足菌株对氧气的需求。此外，还可以通过筛选溶氧相关的蛋白基因，增强菌株在低氧环境下的氧气摄取能力。另一方面，可以对结冷胶合成路径酶基因进行系统的代谢工程改造，通过提高前体的积累、增强四糖单元的组装、促进产物外排等手段来进一步提高菌株整体的生产性能。
作者利益冲突公开声明
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作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
基金
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  • 国家重点研发计划(2023YFA0914300)
文献
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doi: 10.13343/j.cnki.wsxb.20240844
  • 接收时间：2024-12-27
  • 首发时间：2026-02-06
  • 出版时间：2025-07-04
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出版历史
  • 收稿日期：2024-12-27
  • 录用日期：2025-01-27
基金
National Key Research and Development Program of China(2023YFA0914300)
国家重点研发计划(2023YFA0914300)
作者信息
    1.安徽工程大学 生物与食品工程学院，安徽省工业微生物分子育种工程实验室，安徽 芜湖
    2.江南大学 生物工程学院，工业生物技术教育部重点实验室，江苏 无锡
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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20240844
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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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