CN109251938A - 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 - Google Patents
一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 Download PDFInfo
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- CN109251938A CN109251938A CN201710571717.5A CN201710571717A CN109251938A CN 109251938 A CN109251938 A CN 109251938A CN 201710571717 A CN201710571717 A CN 201710571717A CN 109251938 A CN109251938 A CN 109251938A
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- xylose
- acidilactici
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Abstract
本发明公开了一种共发酵葡萄糖和木糖生产L‑乳酸的乳酸片球菌构建方法,属于基因工程技术领域。构建步骤包括,利用热敏型敲除系统在L‑乳酸生产菌株乳酸片球菌Pediococcus acidilactici TY112(保藏号为CGMCC NO.8664)基因组上整合异源木糖异构酶,木酮糖激酶,转酮醇酶以及转醛醇酶编码基因;敲除磷酸转酮酶和乙酸激酶编码基因;适应性进化提高共发酵葡萄糖和木糖的能力。本发明成功得到了一株高效共发酵葡萄糖和木糖生产光学纯L‑乳酸的工程菌株,并命名为P.acidilactici ZY271,其保藏号为CGMCC NO.13611。
Description
技术领域
本发明属于基因工程领域,特别涉及一种基于同源重组原理,利用热敏型敲除系统的共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌构建方法。
背景技术
乳酸是一种重要的工业化学品,它广泛运用于食品、医药、皮革以及纺织业。近年来,以光学纯L-乳酸为前体生产生物可降解性塑料聚乳酸,使得光学纯L-乳酸的需求大大提升。目前,乳酸生产主要包括两种路径:化学合成和微生物发酵。化学合成主要缺点在于其生产的乳酸是D/L混合型乳酸,而微生物发酵优点在于可生产光学纯乳酸、低的生产温度以及低能耗,使得微生物发酵成为主要的乳酸生产方式。世界上90%的商用L-乳酸都是由玉米淀粉类原料进行发酵得到的,而玉米淀粉原料的成本约占总发酵成本的70%。因此,亟需找到一种廉价的原料替代淀粉类原料进行L-乳酸生产。木质纤维素原料是一种价格低廉、来源广泛的可再生生物质能源,利用廉价、含量丰富的木质纤维素原料将会大大减少L-乳酸生产成本。
木质纤维素主要包括纤维素(30-60%干重)、半纤维素(20-40%)和木质素(15-25%)。其中纤维素来源的葡萄糖可以被微生物利用。半纤维来源的木糖约占木质纤维素来源总糖的30%,但大部分生物炼制菌株都不能利用木糖,木糖的不可利用是木质纤维素原料进行高指标生物基化学品生产的一个重要难点。本实验室先前工作中,从玉米秸秆乙醇发酵醪中筛选得到一株乳酸片球菌P.acidilaticiDQ2,该菌可以很好的适应木质纤维素体系,并可以利用玉米秸秆原料获得103g/L的D/L混合型乳酸。之后通过基因工程改造敲除了P.acidilatici DQ2基因组上D-乳酸脱氢酶基因ldhD,得到的工程菌株P.acidilaticiTY112可以利用30%(w/w)固含量的玉米秸秆原料产生104.5g/L的光学纯L-乳酸,但其不能利用木糖。如果将木糖代谢路径构建至P.acidilatici TY112中,共发酵玉米秸秆中的葡萄糖和木糖,L-乳酸的产量必将大大提高。
据文献报道,乳酸菌中木糖代谢路径构建以生产L-乳酸只在Lactococcus lactis中实现过。Shinkawa等(Shinkawa S,Okano K,Yoshida S,Tanaka T,Ogino C,Fukuda H,Kondo A.Improved homo L-lactic acid fermentation from xylose by abolishmentof the phosphoketolase pathway and enhancement of the pentose phosphatepathway in genetically modified xylose-assimilating Lactococcus lactis.ApplMicrobiol Biotechnol,2011,91:1537-1544.)敲除了PK路径的磷酸转酮酶编码基因pkt,并将异源的转酮醇酶编码基因(tkt)整合至pkt基因位点处,并携带了xylRAB的表达质粒,得到的工程菌株可以通过PP路径利用木糖进行同型L-乳酸生产。但是该文献中的xylRAB是在质粒中进行表达的,并没有整合到基因组上,得到的工程菌株并不是一株稳定的工程菌株,在发酵过程中需要添加抗生素维持重组菌的正常生长,抗生素的添加将会增加发酵成本,对于大规模工业运用来说是一个重要缺陷。另外该文献及作者后期的研究中并没有使用木质纤维素原料进行共发酵生产L-乳酸,没有实际证明该工程菌株具备共发酵葡萄糖和木糖生产L-乳酸的能力。
目前没有乳酸片球菌共发酵葡萄糖和木糖生产L-乳酸的报道,因此在乳酸片球菌中构建木糖代谢路径生产L-乳酸,对于共发酵木质纤维素原料中的葡萄糖和木糖进行高浓度L-乳酸生产具有重要现实意义。
发明内容
本发明目的在于构建一株能够高效共发酵葡萄糖和木糖进行L-乳酸生产的P.acidilactici工程菌株。
本发明实现P.acidilactici共发酵葡萄糖和木糖生产L-乳酸所采用的技术方案是:首先,敲除PK路径中的磷酸转酮酶基因pkt以阻断PK路径,从而减少副产物乙酸生成。然后,将异源的磷酸转酮酶(tkt)和转酮醇酶(tal)表达框整合至已敲除的pkt基因位点处,从而引入PP路径。接着,将xylAB表达框整合至乙酸激酶ackA2处,在引入木糖代谢路径的同时,插入失活乙酸激酶基因ackA2。最后,将得到的工程菌株在木糖为唯一碳源的合成培养基中进行适应性进化,直至细胞生长、发酵液中的木糖残留以及L-乳酸生产稳定为止。
上述基因敲除以及基因整合具体方法是:通过电穿孔技术,将构建得到的敲除质粒或整合质粒转化至P.acidilactici中,涂布于含红霉素的MRS平板,28℃静置3天,然后将平板上长出的单菌落接种至含红霉素的液体MRS培养基中,42℃培养16h后,取1微升菌液稀释106倍后,取200微升涂布含红霉素的MRS平板上,42℃静置培养24h。将长出的单菌落接种至不含红霉素的液体MRS培养基中,28℃培养36h后,取1微升菌液稀释106倍后,取200微升涂布于无抗性的MRS平板上,42℃静置培养24h。将长出的单菌落尽可能多的分别点板至不含红霉素的MRS平板和含红霉素的MRS平板上,42℃静置培养24h,将在无红霉素的平板上生长、而含红霉素的平板上不生长的菌落进行进一步的基因组PCR验证,以确认是否完成了基因敲除或基因整合。
上述适应性进化具体方法是:将整合了木糖代谢路径的工程菌株接种至MRS液体培养基中,42℃培养12h后,以10%(v/v)的接种量接种至含40g/L木糖为唯一碳源的MRS液体培养基中,培养24h后,以10%接种量转接至新鲜的木糖MRS液体培养基中继续培养24h(其中进行木糖,L-乳酸含量测定的摇瓶中需添加CaCO3进行pH调节,而进行细胞生长监测的摇瓶中不添加CaCO3)。长期驯化不断进行,直至发酵液中残留的木糖、L-乳酸生成以及细胞生长稳定为止,对得到的木糖发酵性能稳定的驯化菌株进行甘油保种。
本发明结合代谢工程以及适应性进化,首次在P.acidilactici中实现共发酵葡萄糖和木糖生产L-乳酸。
本发明结合代谢工程和适应性进化,得到了一株高效共发酵葡萄糖和木糖进行L-乳酸生产的工程菌株,并命名为P.acidilactici ZY271,该菌株已于2017年01月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC NO.13611,其分类命名为乳酸片球菌(Pediococcusacidilactici)。
附图说明
图1:P.acidilactici中木糖代谢路径构建示意图。
图2:P.acidilactici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的基因组PCR验证图
图中:M,DL5,000DNA Marker;1,利用引物ldhD-F和ldhD-R扩增D-乳酸脱氢酶基因ldhD;2,利用引物ldh-F和ldh-R扩增已敲除的L-乳酸脱氢酶基因ldh;3,利用引物pkt-F和pkt-R扩增已敲除的磷酸转酮酶基因pkt;4,利用引物tkt_tal-F和tkt_tal-R扩增整合至基因组上的异源基因tkt_tal;5,利用引物Em-F和Em-R扩增红霉素抗性基因Em;6,利用引物xylAB_2911-F和xylAB_2911-R扩增异源木糖异构酶和木酮糖激酶基因。
图3:适应性进化过程中的木糖残留、L-乳酸生成以及细胞生长监测。
图4:驯化前后菌株发酵木糖生产L-乳酸性能比较。
图5:驯化前后菌株共发酵葡萄糖和木糖生产L-乳酸性能比较。
图6:木糖利用工程菌株P.acidilactici ZY271与母本菌株P.acidilacticiTY112共发酵葡萄糖和木糖生产L-乳酸性能比较。
具体实施方案
一、本发明所使用的菌株与质粒
L-乳酸生产菌株P.acidilactici TY112是将野生型D/L-乳酸生产菌株P.acidilactici DQ2的D-乳酸脱氢酶基因ldhD敲除得到,已于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC NO.8664。质粒构建所用的宿主菌Escherichia coli XLI-blue保存于本实验室。基因xylAB_2911,tkt和tal来源于乳酸片球菌P.acidilactici DSM20284,购自DSM(Scheiwg,Germany)。所用表达质粒pMG36e来源见Van de Guchte M,Van der Vossen JM,Kok J,Venema G.Construction of alactococcal expression vector:expression of hen egg white lysozyme inLactococcus lactis subsp.lactis.Applied Environment and Microbiology,1989,55(1):224-228。所用热敏型敲除质粒pSET4E来源见Yi X,Zhang P,Sun J,Tu Y,Gao Q,ZhangJ,Bao J.Engineering wild-type robust Pediococcus acidilactici strain for hightiter l-and d-lactic acid production from corn stover feedstock.Journal ofBiotechnology,2016,217:112-121。二、试剂与培养基
DNA聚合酶购自Takara生物工程公司;限制性内切酶和T4DNA ligase购自Fermentas公司;基因组抽提、质粒抽提、PCR产物回收、DNA凝胶回收试剂盒均购自上海捷瑞生物工程有限公司;乳酸光学纯度检测试剂盒购自Megazyme公司;红霉素购自Biosharp生物科技有限公司;酵母粉和蛋白胨购买于Oxoid,Hampshire,UK;基因测序由上海美吉生物医药科技有限公司完成;引物合成由上海捷瑞生物工程有限公司完成。
MRS培养基:1L去离子水中含有20.0g葡萄糖,10.0g蛋白胨,4.0g酵母粉,8.0g牛肉粉,2.0g柠檬酸氢二铵,3.0g无水乙酸钠,2.0g磷酸氢二钾,0.2g七水硫酸镁,0.05g一水硫酸锰和1mL的Tween 80;
简化MRS液体培养基:1L去离子水中含有20.0g葡萄糖,10.0g蛋白胨,10.0g酵母粉,5.0g无水乙酸钠,2.0g柠檬酸氢二铵,2.0g磷酸氢二钾,0.58g七水硫酸镁和0.25g一水硫酸锰;
适应性进化所用培养基:1L去离子水中含有40.0g木糖,10.0g蛋白胨,10.0g酵母粉,5.0g乙酸钠,2.0g柠檬酸氢二铵,2.0g磷酸氢二钾,0.58g七水硫酸镁,0.25一水硫酸锰,12g CaCO3(进行细胞生长的摇瓶不加CaCO3);
LB培养基:
液体:1L去离子水中含有5.0g酵母粉,10.0g蛋白胨,10.0g氯化钠;固体:在液体培养基中额外添加15.0g/L的琼脂;
以上培养基均需115℃高压蒸汽灭菌20min。
三、所用主要仪器
Mastercycler型PCR仪(Eppendorf公司);Gene Pulser Xcell型电穿孔仪(Bio-Rad公司);EPS-100型DNA电泳系统(上海天能公司);Tanon 1600凝胶成像系统(上海天能公司);DU-800型核酸蛋白质分析仪(Beckman公司);LC-20AD型高效液相色谱(岛津公司)。
实施案例1:木糖代谢在P.acidilactici TY112中的实现。
(1)表达质粒pMG36e-PldhD-xylAB_2911的构建
首先以P.acidilactici DSM20284基因组为模板,以xylAB_2911-F(SEQ ID NO:1)和xylAB_2911-R(SEQ ID NO:2)为引物扩增得到xylAB_2911,以P.acidilactici TY112为模板,以PldhD-F(SEQ ID NO:3)和PldhD-R(SEQ ID NO:4)为引物扩增得到ldhD起始密码子上游约300bp的启动子序列PldhD,然后通过融合PCR技术得到表达框PldhD_xylAB_2911,然后通过EcoR I和Xba I双酶切,用表达框PldhD_xylAB_2911替换pMG36e自身的P32启动子,得到表达质粒pMG36e-PldhD_xylAB_2911。
(2)重组菌P.acidilactici TY112(pMG36e-PldhD_xylAB_2911)的木糖发酵能力验证
将(1)中得到的表达质粒pMG36e-PldhD_xylAB_2911通过电穿孔技术转化至P.acidilactici TY112中,得到重组菌P.acidilactici TY112(pMG36e-PldhD_xylAB_2911)。该重组菌可利用木糖进行生长,48h可以消耗12.61g/L木糖,产生5.62g/L的L-乳酸,但同时产生了6.67g/L的副产物乙酸。这说明重组菌可以通过PK路径代谢木糖生产L-乳酸,并伴随着大量副产物乙酸生成(见表1)。
实施案例2:磷酸转酮酶路径(PK路径)的阻断
(1)敲除质粒pSET4E-Δpkt的构建
以P.acidilactici TY112基因组为模板,以up-pkt-F(SEQ ID NO:5)和up-pkt-R(SEQ ID NO:6)为引物扩增得到pkt基因的上游约1,000bp同源臂片段(up-pkt),以down-pkt-F(SEQ ID NO:7)和down-pkt-R(SEQ ID NO:8)为引物扩增得到pkt基因的下游约1,000bp同源臂片段序列(down-pkt)。将down-pkt片段插入至pSET4E的BamH I和Sac I位点之间,然后将up-pkt片段插入至Pst I和Sal I位点之间,得到用于pkt基因敲除的质粒pSET4E-Δpkt。
(2)磷酸转酮酶基因pkt的敲除
将敲除质粒pSET4E-Δpkt电转化至P.acidilactici TY112中,得到重组菌P.acidilactici TY112(pSET4E-Δpkt),将重组菌接种至含红霉素的液体MRS培养液中,42℃培养16h后,取1微升菌液稀释106倍后,取200微升涂布含红霉素的MRS平板上,42℃静置培养24h。将长出的单菌落接种至不含红霉素的液体MRS培养液中,28℃培养36h后,取1微升菌液稀释106倍后,取200微升涂布于无抗性的MRS平板上,42℃静置培养24h。将长出的单菌落尽可能多的分别点板至不含红霉素的MRS平板和含红霉素的MRS平板上,42℃静置培养24h,将在无红霉素的平板上生长、而含红霉素的平板上不生长的菌落进行进一步的基因组PCR验证。以pkt-F(SEQ ID NO:9)和pkt-R(SEQ ID NO:10)为引物扩增pkt,若扩增不出磷酸转酮酶基因pkt,则基因pkt被敲除,得到的工程菌株命名为P.acidilactici TY112-Δpkt。
(3)工程菌P.acidilactici TY112-Δpkt(pMG36e-PldhD-xylAB_2911)的木糖发酵
将表达质粒pMG36e-PldhD_xylAB_2911电转化至工程菌株P.acidilacticiTY112-Δpkt中,得到重组菌P.acidilactici TY112-Δpkt(pMG36e-PldhD_xylAB_2911),将重组菌培养在含35g/L木糖的MRS液体培养基中。该重组菌的木糖利用能力显著下降,48h内只消耗了4.90g/L的木糖,只产生了0.19g/L的L-乳酸,而乙酸的生成也降至0.20g/L(见表1)。这说明pkt的敲除成功阻断了PK路径,使得菌株基本不能生成副产物乙酸,但不生成L-乳酸说明P.acidilactici TY112中只存在PK路径进行L-乳酸生产。
实施案例3:戊糖磷酸路径(PP路径)的构建
(1)整合质粒pSET4E-Δpkt::(tkt_tal)的构建
以P.acidilactici TY112基因组为模板,以PldhD-F(SEQ ID NO:3)和PldhD-R(SEQ ID NO:4)为引物扩增得到启动子PldhD;以P.acidilactici DSM20284为模板,设计引物tkt_tal-F(SEQ ID NO:11)和tkt_tal-R(SEQ ID NO:12)扩增得到tkt_tal。接着将克隆得到的PldhD与tkt_tal通过融合PCR得到表达框PldhD_tkt_tal,并插入至案例2得到的敲除质粒pSET4E-Δpkt的Sal I和BamH I之间,得到整合质粒pSET4E-Δpkt::(tkt_tal)。
(2)tkt_tal的基因组整合
将整合质粒pSET4E-Δpkt::(tkt_tal)电转化至案例2得到的工程菌株P.acidilactici TY112-Δpkt中,得到重组菌P.acidilactici TY112-Δpkt(pSET4E-Δpkt::(tkt_tal))。通过与上述类似的基因敲除与整合方法,筛选得到的单菌落为模板,若能扩增出tkt_tal,而扩增不出红霉素抗性基因Em,则基因tkt_tal基因组整合成功,得到的工程菌株命名为P.acidilatici TY112-Δpkt::(tkt_tal)。
(3)重组菌P.acidilatici TY112-Δpkt::(tkt_tal)(pMG36e-PldhD_xylAB_2911)的木糖发酵
将表达质粒pMG36e-PldhD_xylAB_2911通过电穿孔技术转化至P.acidilaticiTY112-Δpkt::(tkt_tal),将得到的重组菌P.acidilatici TY112-Δpkt::(tkt_tal)(pMG36e-PldhD_xylAB_2911)接种于简化MRS液体培养基中,培养12h后,以10%接种量转接至含35g/L木糖的MRS液体培养基中进行木糖发酵。工程菌株在48h内可以消耗9.64g/L木糖,产生了5.86g/L的L-乳酸,但同时产生了1.73g/L的乙酸(见表1)。这一结果说明异源的tkt_tal成功在该菌株中表达,并且tkt_tal的整合,成功将PP路径引入到工程菌株中,使得菌株又恢复了代谢木糖产L-乳酸的能力,但仍有少量乙酸产生。
实施案例4:xylAB表达框的整合以及ackA2基因的插入失活
(1)整合质粒pSET4E-ΔackA2::xylAB的构建
首先以P.acidilactici TY112基因组为模板,以up-ackA2-F(SEQ ID NO:13)和up-ackA-R(SEQ ID NO:14)为引物扩增得到ackA2上游约700bp的同源臂序列up-ackA2,以down-ackA2-F(SEQ ID NO:15)和down-ackA-R(SEQ ID NO:16)为引物扩增扩增得到ackA2下游约700bp的同源臂序列down-ackA2。以表达质粒pMG36e-PldhD_xylAB_2911为模板,以PldhD_xylAB_2911-F(SEQ ID NO:17)和PldhD_xylAB_2911-R(SEQ ID NO:18)为引物扩增得到PldhD_xylAB_2911。然后通过酶切连接,将up-ackA2片段插入至pSET4E的Hind III和BamH I位点之间,将down-ackA2片段插入至BamH I和EcoR I位点之间,得到用于ackA2基因缺失的质粒pSET4E-ΔackA2,然后将表达框PldhD_xylAB_2911插入至pSET4E-ΔackA2的Xho I和BamH I之间,得到用于将ackA2失活,同时将xylAB_2911整合至ackA2基因内部的质粒pSET4E-ΔackA2::xylAB。
(2)xylAB的基因组整合以及ackA2基因的插入失活
将整合质粒pSET4E-ΔackA2::xylAB电转化至P.acidilatici TY112-Δpkt::(tkt_tal)中,得到重组菌P.acidilatici TY112-Δpkt::(tkt_tal)(pSET4E-ΔackA2::xylAB),通过与上述类似的基因敲除与整合方法,筛选得到的单菌落为模板,若能够扩增出xylAB,而扩增不出红霉素抗性基因Em,则xylAB整合成功,得到的整合菌株命名为P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB。
(3)工程菌P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的木糖发酵
将工程菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB接种于简化MRS液体培养基中,培养12h后,以10%接种量转接至含35g/L木糖的MRS液体培养基中进行木糖发酵。工程菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB可以代谢木糖生产L-乳酸,但木糖代谢速率较慢(见表1)。
表1:P.acidilatici TY112相关工程菌株的木糖发酵性能比较
实施案例5:工程菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的适应性进化
案例4中得到的工程菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB木糖代谢速率很慢。本发明采取了适应性进化策略提高该工程菌株的木糖代谢速率。首先,将工程菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB接种至MRS液体培养基中,培养12h后,以10%的接种量接种至以木糖为唯一碳源的MRS液体培养基中(添加CaCO3调节pH),培养24h后,以10%接种量转接至新鲜含木糖为唯一碳源的MRS液体培养基中继续培养24h(用于检测细胞生长的摇瓶中不添加CaCO3调节pH,其余条件一样),期间不断进行转接,直至细胞生长、发酵液中残留的木糖以及L-乳酸生成稳定为止。连续转接66次(66天)后,驯化菌株的木糖发酵性能有了显著提高,且保持稳定(见图3),对得到的木糖发酵性能稳定的驯化菌株进行保种,并命名为P.acidilactici ZY271。
实施案例6:驯化前后木糖发酵性能比较
(1)木糖为唯一碳源的发酵性能比较
将驯化菌株P.acidilactici ZY271与未驯化菌株P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB进行木糖为唯一碳源的发酵性能比较。将-80℃冰箱保存的两株菌株分别接种至简化MRS液体培养基中活化12h后,以10%接种量分别接种至含35g/L木糖为唯一碳源的MRS培养基中进行发酵性能比较(添加CaCO3进行pH调节),用于检测细胞生长的摇瓶中不添加CaCO3。
就生长而言,在培养16h后,驯化菌株P.acidilactici ZY271的OD600达到了4.50,远高于未驯化菌株的1.74。就木糖发酵能力而言,驯化菌株的木糖消耗速率,L-乳酸生产速率均高于未驯化菌株(见图4)。长期驯化大大提升了工程菌株利用木糖的能力。
(2)葡萄糖和木糖的共发酵性能比较
接着又比较了驯化菌株和未驯化菌株的葡萄糖木糖共发酵能力。使用的培养基是含有40g/L葡萄糖和40g/L木糖的MRS培养基。两菌株的细胞生长以及葡萄糖消耗没有什么差别,但驯化菌株P.acidilactici ZY271在48h内90.0%的木糖被消耗,而未驯化菌株只消耗了58.0%的木糖(见图5)。这说明,适应性进化也大大提升了工程菌株的共发酵能力。依据Megazyme D-/L-Lactic acid Kit试剂盒方法,测得L-乳酸光学纯度为99.6%。
实施案例7:木糖利用工程菌株与母本菌株的共发酵性能比较
将本发明构建的木糖利用工程菌株P.acidilactici ZY271和不能利用木糖的母本菌株P.acidilactici TY112分别接种至简化MRS液体培养基中活化12h后,以10%接种量分别接种至含40g/L葡萄糖和40g/L木糖的MRS培养基中进行共发酵性能比较(添加CaCO3进行pH调节)。工程菌株P.acidilactici ZY15在48h已基本耗完所有葡萄糖和木糖;而母本菌株只消耗了葡萄糖,木糖并没有消耗(见图6)。以上结果说明,本发明通过代谢工程和适应性进化成功构建了一株具备共发酵葡萄糖和木糖能力的L-乳酸生产菌株。
以上具体描述了本发明技术方案的操作实例,不视为对本发明的应用限制。凡操作条件的等同替换,均在本发明的保护范围之内。
序列表
<110> 华东理工大学
<120> 一种共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌构建方法
<130> 2017-06-01
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> xylAB_2911-F
<400> 1
aaaagaaagg ggtaatatta caatgtcatt atttgatcgc aaaaaaatgg 50
<210> 2
<211> 35
<212> DNA
<213> xylAB_2911-R
<400> 2
ctagtctaga ttacaacatt tcacgccggt aattc 35
<210> 3
<211> 31
<212> DNA
<213> PldhD-F
<400> 3
ccggaattct gctctggtgt gcagaccaga c 31
<210> 4
<211> 49
<212> DNA
<213> PldhD-R
<400> 4
ttttttgcga tcaaataatg acattgtaat attacccctt tctttttta 49
<210> 5
<211> 31
<212> DNA
<213> up-pkt-F
<400> 5
aactgcagcc aagtggttcg tgaatttgtt g 31
<210> 6
<211> 37
<212> DNA
<213> up-pkt-R
<400> 6
gcgtcgactt aaagcttaaa gattaaacaa taaaagc 37
<210> 7
<211> 31
<212> DNA
<213> down-pkt-F
<400> 7
cgcggatccc gtaatcttcc tttcgtgagc g 31
<210> 8
<211> 32
<212> DNA
<213> down-pkt-R
<400> 8
cgagctcagc aatgtatttt agcacattga gc 32
<210> 9
<211> 29
<212> DNA
<213> pkt-F
<400> 9
atgacagact attcatctaa agcttactt 29
<210> 10
<211> 25
<212> DNA
<213> pkt-R
<400> 10
ttatttaacg tctttccata cccag 25
<210> 11
<211> 46
<212> DNA
<213> tkt_tal-F
<400> 11
aaagaaaggg gtaatattac agtgtttact aaaaaagata ttgacg 46
<210> 12
<211> 32
<212> DNA
<213> tkt_tal-R
<400> 12
aactgcagct ataaaattga caatcccgat tc 32
<210> 13
<211> 29
<212> DNA
<213> up-ackA2-F
<400> 13
cccaagcttt tcgcgaattt gttaacgct 29
<210> 14
<211> 42
<212> DNA
<213> up-ackA2-R
<400> 14
cgcggatccg cgccgctcga gccaaatggc aggtgattaa tt 42
<210> 15
<211> 32
<212> DNA
<213> down-ackA2-F
<400> 15
cgcggatcct tagcgtagaa gaagtcgttg ac 32
<210> 16
<211> 30
<212> DNA
<213> down-ackA2-R
<400> 16
ccggaattca gacaaaacca aaagagcgtg 30
<210> 17
<211> 31
<212> DNA
<213> PldhD-xylAB_2911-F
<400> 17
ccgctcgagt gctctggtgt gcagaccaga c 31
<210> 18
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<212> DNA
<213> PldhD-xylAB_2911-R
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Claims (6)
1.一种共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌构建方法,步骤包括:
(1)木糖代谢在P.acidilactici TY112中的实现:将携带木糖异构酶(xylA)和木酮糖激酶(xylB)的表达质粒pMG36e-PldhD_xylAB_2911电转化至P.acidilactici TY112,得到的重组菌P.acidilactici TY112(pMG36e-PldhD_xylAB_2911)可利用木糖进行生长和L-乳酸生产,但同时产生了大量副产物乙酸。
(2)磷酸转酮酶路径(PK路径)的阻断:通过同源重组敲除P.acidilactici TY112基因组上的磷酸转酮酶基因pkt,以阻断PK路径从而减少副产物乙酸生成,得到的工程菌株为P.acidilactici TY112-Δpkt。
(3)戊糖磷酸路径(PP路径)的构建:在步骤(2)得到的菌株基础上,通过同源重组将异源的转酮醇酶基因(tkt)和转醛醇酶基因(tal)整合至步骤(2)中敲除的pkt基因位点处,使菌株通过PP路径代谢木糖,得到的工程菌株为P.acidilactici TY112-Δpkt::(tkt_tal)。
(4)xylAB的整合以及乙酸激酶ackA2的插入失活:在步骤(3)得到的菌株基础上,将步骤(1)中的xylAB表达框整合至乙酸激酶ackA2位点处,同时将ackA2进行插入失活,得到的工程菌株为P.acidilactici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB。
(5)适应性进化加快木糖代谢:将步骤(4)得到的工程菌株,在木糖为唯一碳源的培养基中进行适应性进化以提高木糖代谢能力。
2.根据权利要求书1,其特征在于,步骤(1)所使用的表达质粒pMG36e-PldhD_xylAB_2911中,其启动子PldhD是P.acidilactici TY112基因组上D-乳酸脱氢酶编码基因(ldhD)起始密码子上游约300bp的启动子序列,xylAB_2911来源于P.acidilactici DSM20284。
3.根据权利要求书1,其特征在于,步骤(3)中使用的tkt和tal基因来源于P.acidilactici DSM 20284,所用启动子是PldhD。
4.根据权利要求书1,其特征在于,步骤(5)中采用的适应性进化方法是:将步骤(4)得到的工程菌株P.acidilactici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB在简化MRS培养基中培养12h后,以10%(v/v)的接种量接种至以40g/L木糖为唯一碳源的MRS培养基中,之后,每24h以10%接种量转接至新鲜的木糖MRS培养基中进行发酵,直至细胞生长、发酵液中残留的木糖以及生成的L-乳酸稳定时停止驯化。
5.根据权利要求书4,其特征在于,经过66次的连续转接(共驯化66天),得到的木糖发酵性能稳定的驯化菌株命名为P.acidilactici ZY271,其保藏编号为CGMCC NO.13611。
6.根据权利要求书5,其特征在于,P.acidilactici ZY271能高效利用木糖生产L-乳酸,并能高效共发酵葡萄糖和木糖生产高纯度L-乳酸,L-乳酸纯度高达99.6%。
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