CN115094016B - 敲除葡萄糖-6-磷酸异构酶基因的重组大肠杆菌及其在生产1,2,4-丁三醇中的应用 - Google Patents
敲除葡萄糖-6-磷酸异构酶基因的重组大肠杆菌及其在生产1,2,4-丁三醇中的应用 Download PDFInfo
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Abstract
本发明属于微生物工程和发酵技术领域,具体涉及敲除葡萄糖‑6‑磷酸异构酶基因的重组大肠杆菌及其在生产1,2,4‑丁三醇中的应用。本发明以大肠杆菌为出发菌株,通过敲除木糖异构酶xylA、2‑酮基‑3‑脱氧木糖酸醛缩酶基因yjhH和yagE、木糖酸操纵子转录抑制子基因xynR、特异性葡萄糖转运蛋白基因ptsG和葡萄糖‑6‑磷酸异构酶编码基因pgi;同时敲入木糖酸脱水酶基因xylD、2‑酮酸脱羧酶基因kdcA和木糖脱氢酶及木糖酸内酯酶编码基因xylBC获得,得到的工程大肠杆菌E.coli 4KI03能够以玉米芯水解液为底物高效生产1,2,4‑丁三醇,具有良好的实际应用之价值。
Description
技术领域
本发明属于微生物工程和发酵技术领域,具体涉及敲除葡萄糖-6-磷酸异构酶基因的重组大肠杆菌及其在生产1,2,4-丁三醇中的应用,具体的,所述应用为以木糖或玉米芯水解液为底物高效发酵生产1,2,4-丁三醇。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
1,2,4-丁三醇(1,2,4-butanetriol,简称BT)是一种无色无臭无毒、有甜味、呈粘稠状的水溶性C4化合物,分子式为C4H10O3。1,2,4-丁三醇可作为重要的中间体合成多种有机化合物,在军事、制药、烟草、服装、造纸、化妆品、造纸、喷墨及高分子材料等不同领域具有广泛应用。例如,在军事领域,1,2,4-丁三醇最重要的用途是用于合成丁三醇三硝酸酯(简称BTTN)。与硝化甘油相比,BTTN具有冲击敏感性低、能量水平高、热稳定性高、低温不脆变、反应安全性高等优点,是硝化甘油的优良替代品。在医药领域,1,2,4-丁三醇可用于合成降胆固醇药物、抗癌药物以及作为药物缓释剂增加患者的适应性、降低药物副作用等。在烟草行业,卷烟中添加1,2,4-丁三醇可以减轻硝基化合物对人体的危害,降低毒性。在高分子材料领域,1,2,4-丁三醇可用于合成聚氨酯泡沫。另外,1,2,4-丁三醇还可添加在彩色显影液中,用来增加成像的稳定性和色彩饱和度。
生物合成法具有反应条件温和、安全性高、原料来源广泛等优点。目前,已报道的生物法合成1,2,4-丁三醇的菌株主要有大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)、肺炎克雷伯氏菌(Klebsiella pneumoniae),底物主要有葡萄糖、木糖、阿拉伯糖及苹果酸等。自然界中不存在1,2,4-丁三醇的天然生物合成途径,因此,1,2,4-丁三醇的生物合成需要对不同来源的生物途径进行组合与优化。以木糖为底物的1,2,4-丁三醇合成途径主要通过木糖脱氢、木糖酸脱水、酮基木糖酸脱羧及醇脱氢四步催化反应完成。2014年,Valdehuesa等人阻断木糖和2-酮基-3-脱氧木糖酸的内源代谢途径,利用双质粒分别表达木糖脱氢酶编码基因xdh与苯甲酰甲酸脱羧酶编码基因mdlC,利用10g/L木糖生成0.88g/L的1,2,4-丁三醇,转化率为12.86%,首次实现了以木糖为底物基于单一微生物的1,2,4-丁三醇合成。江南大学Jing等筛选并在大肠杆菌中表达高效的脱羧酶KivD,阻断分支途径,增强1,2,4-丁三醇合成途径的碳通量,将1,2,4-丁三醇产量提高至10.03g/L。2021年,狄莹莹等人弱化乙酸合成途径,在5L发酵罐中1,2,4-丁三醇最高产量为16.1g/L。
目前,1,2,4-丁三醇高产菌株构建主要围绕提高木糖的代谢通量、筛选高效的α-酮酸脱羧酶、阻断分支途径、弱化乙酸合成途径、发酵条件优化等策略展开,但发明人发现,1,2,4-丁三醇产量及生产效率仍处于较低水平,难以满足日益增长的工业需求。
发明内容
针对上述发酵生产1,2,4-丁三醇的技术中,存在的生产成本高,产量低,生产效率低,难以满足工业化生产等不足,发明人经长期的技术与实践探索,提供一株重组大肠杆菌及其在利用木糖发酵生产1,2,4-丁三醇中的应用。本发明基于代谢工程的改造获得一株重组大肠杆菌,其能够利用木糖高效发酵生产1,2,4-丁三醇,解除了1,2,4-丁三醇生产过程中辅因子失衡的限制因素,为解决木糖高效利用等问题提供了途径。基于上述研究成果,从而完成本发明。
为实现上述技术目的,本发明采用如下技术方案:
本发明的第一个方面,提供一株重组大肠杆菌,所述重组大肠杆菌是以大肠杆菌为出发菌株,通过敲除木糖异构酶xylA、2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE、木糖酸操纵子转录抑制子基因xynR、特异性葡萄糖转运蛋白基因ptsG和葡萄糖-6-磷酸异构酶编码基因pgi;同时在基因组xynR位点敲入木糖酸脱水酶基因xylD和2-酮酸脱羧酶基因kdcA,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC获得。该重组大肠杆菌菌株其基因型为E.coli W3110(DE3)ΔxylA::xylBCΔyjhHΔyagEΔxynR::xylD﹠kdcAΔpgi,命名为E.coli 4KI03。
其中,所述出发菌株具体为E.coli W3110(DE3),该菌株可通过市售方式购得。
所述木糖异构酶xylA的核苷酸序列如SEQ ID NO.1所示。
所述2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
所述木糖酸操纵子转录抑制子基因xynR的核苷酸序列如SEQ ID NO.4所示。
来源于月柄杆菌(Caulobacter crescentus)的木糖酸脱水酶基因xylD和来源于乳酸乳球菌(Lactococcus lactis)的2-酮酸脱羧酶基因kdcA的核苷酸序列分别如SEQ IDNO.5和SEQ ID NO.6所示。
所述特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi的核苷酸序列如SEQ ID NO.7和SEQ ID NO.9所示。
所述木糖脱氢酶及木糖酸内酯酶编码基因xylBC来源于月柄杆菌(Caulobactercrescentus),其核苷酸序列如SEQ ID NO.8所示。
本发明的第二个方面,提供上述重组大肠杆菌的构建方法,所述构建方法包括:敲除出发菌株大肠杆菌木糖异构酶xylA;敲除2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE;敲除木糖酸操纵子转录抑制子基因xynR;同时将木糖酸脱水酶基因xylD和的2-酮酸脱羧酶基因kdcA敲入至基因组原xynR位点,敲除其特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC。
本发明的第三个方面,提供一种菌剂,所述菌剂含有上述重组大肠杆菌。
所述菌剂中,除含活性成分外,还含有载体。所述载体可为微生物制剂领域常用的且在生物学上是惰性的载体。
本发明的第四个方面,提供上述重组大肠杆菌或上述菌剂在以木糖或玉米芯水解液为底物发酵生产1,2,4-丁三醇中的应用。
本发明的第五个方面,提供一种发酵生产1,2,4-丁三醇的方法,所述方法包括:以木糖或玉米芯水解液为底物,生物发酵上述重组大肠杆菌,由发酵液得到1,2,4-丁三醇。
与现有技术方案相比,上述一个或多个技术方案具有如下有益效果:
(1)上述技术方案提供的基因工程菌Escherichia coli 4KI03是一株能够高产1,2,4-丁三醇的菌株,其基因敲入了来源于月柄杆菌(Caulobacter crescentus)的木糖酸脱水酶基因xylD和来源于乳酸乳球菌(Lactococcus lactis)的2-酮酸脱羧酶基因kdcA,实现木糖酸到3,4-二羟基丁醛的转化,基因敲入了来自月柄杆菌(Caulobacter crescentus)中木糖脱氢酶基因xylB及木糖酸内酯酶基因xylC,实现了大肠杆菌胞内木糖到木糖酸的转化通过基因敲除阻断木糖和2-酮基-3-脱氧木糖酸的下游代谢途径,阻断葡萄糖磷酸转移酶系统,消除了碳源代谢阻遏,增强了葡萄糖提供的NADPH水平,最终实现了1,2,4-丁三醇高产。
(2)上述技术方案提供的菌株的培养基简单、发酵过程简单、无IPTG诱导、无需添加抗生素、成本低,并且适合工业化应用。
(3)上述技术方案能够发酵生产得到43.4g/L 1,2,4-丁三醇,并具有高得率,高生产效率的优良特点,据申请人所知,其产量是目前生物法生产1,2,4-丁三醇的最高产量,为解决木糖高效利用问题提供了途径。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例中敲除大肠杆菌葡萄糖-6-磷酸异构酶基因对1,2,4-丁三醇生产的影响。
图2为本发明实施例中Escherichia coli 4KI03以木糖为底物的补料分批发酵过程曲线。
图3为本发明实施例中Escherichia coli 4KI03以玉米芯水解液为底物的补料分批发酵过程曲线。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
如前所述,采用生物发酵法生产1,2,4-丁三醇的产量及生产效率仍处于较低水平,难以满足日益增长的工业需求
而细胞代谢往往伴随着氧化还原反应过程,氧化还原酶有不同的辅因子依赖性。辅因子既可以调节胞内氧化还原环境,又可直接作为底物参与目标产物的合成。发明人经研究发现,1,2,4-丁三醇合成途径中,XylBC催化木糖生成木糖酸的过程产生一分子NADH,但催化3,4-二羟基丁醛生成1,2,4-丁三醇的醇脱氢酶YqhD依赖NADPH,存在辅因子不平衡现象,推测这是1,2,4-丁三醇无法进一步提高的限制因素。
因此,迫切需要开发出一种改善胞内辅因子平衡,提高1,2,4-丁三醇产量的方法,既要解决1,2,4-丁三醇生产效率低,产量低的问题,又为其他高值化合物的代谢工程改造提供解决思路。
有鉴于此,本发明的一个典型具体实施方式中,提供一株重组大肠杆菌,所述重组大肠杆菌是以E.coli W3110(DE3)为出发菌株,通过敲除木糖异构酶xylA、2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE、木糖酸操纵子转录抑制子基因xynR、特异性葡萄糖转运蛋白基因ptsG和葡萄糖-6-磷酸异构酶编码基因pgi;同时在基因组xynR位点敲入木糖酸脱水酶基因xylD和2-酮酸脱羧酶基因kdcA,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC获得。
重组大肠杆菌菌株其基因型为E.coli W3110(DE3)ΔxylA::xylBCΔyjhHΔyagEΔxynR::xylD﹠kdcAΔpgi,命名为E.coli 4KI03。
其中,通过在大肠杆菌E.coli W3110(DE3)中基因敲除木糖异构酶xylA,2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE,木糖酸操纵子转录抑制子基因xynR,阻断木糖和2-酮基-3-脱氧木糖酸的下游代谢途径,通过基因敲入将来源于月柄杆菌(Caulobactercrescentus)的木糖酸脱水酶基因xylD和来源于乳酸乳球菌(Lactococcus lactis)的2-酮酸脱羧酶基因kdcA插入商业化质粒pACYCDuet,PCR扩增基因表达框,组建操纵子并敲入至基因组原xynR位点,实现木糖酸到3,4-二羟基丁醛的转化,敲除特异性葡萄糖转运蛋白基因ptsG,阻断磷酸转移酶系统,解除碳源代谢阻遏,增强木糖的转运及利用;通过敲入来源于月柄杆菌(Caulobacter crescentus)的木糖脱氢酶基因xylB与木糖酸脱水酶基因xylC,实现木糖到木糖酸的转化,通过敲除葡萄糖-6-磷酸异构酶编码基因pgi,增强NADPH供给,从而最终获得能够以木糖为底物高效发酵生产1,2,4-丁三醇的重组大肠杆菌。
本发明的又一具体实施方式中,所述木糖异构酶xylA的核苷酸序列如SEQ IDNO.1所示。
本发明的又一具体实施方式中,所述2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
本发明的又一具体实施方式中,所述木糖酸操纵子转录抑制子基因xynR的核苷酸序列如SEQ ID NO.4所示。
本发明的又一具体实施方式中,所述木糖酸脱水酶基因xylD来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.5所示;2-酮酸脱羧酶基因kdcA来源于乳酸乳球菌(Lactococcus lactis),其核苷酸序列如SEQ ID NO.6所示。
本发明的又一具体实施方式中,所述特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi的核苷酸序列如SEQ ID NO.7和SEQ ID NO.9所示。
本发明的又一具体实施方式中,所述木糖脱氢酶编码基因及木糖酸内酯酶编码基因xylBC来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.8所示。
本发明的又一具体实施方式中,提供上述重组大肠杆菌的构建方法,所述构建方法包括:敲除出发菌株E.coli W3110(DE3)的木糖异构酶xylA;敲除2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE;敲除木糖酸操纵子转录抑制子基因xynR;同时将木糖酸脱水酶基因xylD和2-酮酸脱羧酶基因kdcA插入商业化质粒pACYCDuet,PCR扩增基因表达框,组建操纵子并敲入至基因组原xynR位点,敲除其特异性葡萄糖转运蛋白基因ptsG,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC,以及敲除葡萄糖-6-磷酸异构酶编码基因pgi。
本发明的又一具体实施方式中,所述木糖异构酶xylA的核苷酸序列如SEQ IDNO.1所示。
本发明的又一具体实施方式中,所述2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
本发明的又一具体实施方式中,所述木糖酸操纵子转录抑制子基因xynR的核苷酸序列如SEQ ID NO.4所示。
本发明的又一具体实施方式中,所述木糖酸脱水酶基因xylD来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.5所示;2-酮酸脱羧酶基因kdcA来源于乳酸乳球菌(Lactococcus lactis),其核苷酸序列如SEQ ID NO.6所示。
本发明的又一具体实施方式中,所述特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi的核苷酸序列如SEQ ID NO.7和SEQ ID NO.9所示。
本发明的又一具体实施方式中,所述木糖脱氢酶及木糖酸内酯酶编码基因xylBC来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.8所示。
本发明的又一具体实施方式中,上述构建方法中,基因敲除方法为一步法敲除技术,所用的核苷酸突变片段具有敲除基因上下游两个同源臂及卡那霉素抗性基因盒。
本发明的又一具体实施方式中,所述xylA、ptsG和pgi基因的核苷酸突变片段通过PCR直接扩增获得;所述yagE、yjhH的基因敲除,xylD、kdcA和xylBC基因敲入的核苷酸突变片段则通过重组PCR获得。
本发明的又一具体实施方式中,其中扩增核苷酸突变片段的引物序列如SEQ IDNO.10-43所示。
本发明的又一具体实施方式中,提供一种菌剂,所述菌剂含有上述重组大肠杆菌。
所述菌剂中,除含活性成分外,还含有载体。所述载体可为微生物制剂领域常用的且在生物学上是惰性的载体。
所述载体可为固体载体或液体载体;
所述固体载体可为矿物材料、植物材料和/或高分子化合物;所述矿物材料可为粘土、滑石、麦饭石、高岭土、蒙脱石、白碳、沸石、硅石和硅藻土中的至少一种;所述植物材料可为玉米粉、豆粉、稻壳粉和淀粉中的至少一种;所述高分子化合物可为聚乙烯醇或/和聚二醇;
所述液体载体可为有机溶剂、植物油、矿物油或水;所述有机溶剂可为癸烷或/和十二烷。
所述菌剂的剂型可为多种剂型,如液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂;优选为粉剂。
根据需要,所述菌剂中还可添加表面活性剂(如吐温20、吐温80等)、粘合剂、稳定剂(如抗氧化剂)、pH调节剂等。
本发明的又一具体实施方式中,提供上述重组大肠杆菌或上述菌剂在以木糖或玉米芯水解液为底物发酵生产1,2,4-丁三醇中的应用。
本发明的又一具体实施方式中,提供一种发酵生产1,2,4-丁三醇的方法,所述方法包括:以木糖或玉米芯水解液为底物,生物发酵上述重组大肠杆菌,由发酵液得到1,2,4-丁三醇。
其中,所述发酵条件是:培养温度为30±1℃,培养方式为搅拌培养,搅拌转速为400±50转/分钟,通气量为1.5±0.1vvm,调节pH至7.0±0.4,培养时间为36~48小时;
以木糖为底物时,木糖与葡萄糖的含量比例为2-6:1(优选为3:1),木糖含量不低于30g/L;不使用IPTG诱导,使用乳糖进行诱导,乳糖诱导使用浓度优选为10g/L。
本发明的又一具体实施方式中,木糖与葡萄糖的浓度优选为:木糖30g/L;葡萄糖10g/L。
以玉米芯水解液为底物时,玉米芯水解液成分中木糖与葡萄糖的含量比例在6:1~12:1(优选为10:1),木糖含量不低于30g/L;不使用IPTG诱导,使用乳糖进行诱导,乳糖诱导使用浓度优选为10g/L。
本发明的又一具体实施方式中,玉米芯水解液成分优选为:木糖118.5g/L;葡萄糖11.5g/L;阿拉伯糖11.8g/L;甲酸1.4g/L;乙醇0.83g/L;乙酸0.34g/L;糠醛13.5ppm。
经实验证实,本发明的发酵方法以木糖与葡萄糖为底物,能够生产获得1,2,4-丁三醇36.63g/L,生产效率为1.14g/[L·h];该菌以玉米芯水解液,能够生产1,2,4-丁三醇43.4g/L,生产效率为1.09g/[L·h]。本申请重组菌解除了1,2,4-丁三醇生产过程中辅因子失衡的限制因素,从而为工业化生产1,2,4-丁三醇并充分利用木糖提供了新途径。
以下结合具体实例对本发明作进一步的说明,以下实例仅是为了解释本发明,并不对其内容进行限定。凡是依据本发明的技术实质对实施方式所做的任何简单修改,等同变化与修饰,均属于本发明技术方案的范围内。
下述实施例中,所使用的材料、试剂、质粒、专用试剂盒、菌株等,如无特殊说明,均从商业途径得到。
实施例1:Escherichia coli 4KI、Escherichia coli 4KI01、Escherichia coli4KI-P1、Escherichia coli 4KI01-P1、Escherichia coli 4KI02、Escherichia coli4KI03菌株的构建
以大肠杆菌W3110(DE3)为出发菌株,采用Red重组技术(Datsenko KA et al.,Proc.Natl.Acad.Sci.USA.,2000,97:6640-6645)对该菌株进行连续基因改造,大体步骤如下:
(1)采用基因工程手段在大肠杆菌W3110(DE3)中敲除木糖异构酶基因xylA和2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE,阻断菌株利用木糖和木糖酸的内源性途径;敲除木糖酸操纵子转录抑制因子基因xynR,阻断转录抑制因子XynR对木糖酸操纵子的调控,增强木糖酸脱水酶及木糖酸转运蛋白的表达,加强目的产物合成代谢;将来源于月柄杆菌(Caulobacter crescentus)的木糖酸脱水酶基因xylD和来源于乳酸乳球菌(Lactococcuslactis)的支链2-酮酸脱羧酶基因kdcA插入商业化质粒pACYCDuet-1,PCR扩增基因表达框,组建操纵子并敲入至基因组原xynR位点,构建得到工程大肠杆菌菌株Escherichia coli4KI。
(2)将实验室前期构建的pETPtac-xylBC质粒(具体构建方法参见ZHANG Y,GUO S,WANG Y,et al.Production of D-xylonate from corn cob hydrolysate by ametabolically engineered Escherichia coli strain[J].ACS Sustainable Chemistryand Engineering,2019,7(2):2160-2168.)经热激转化法导入宿主Escherichia coli 4KI中得到工程菌株,命名为Escherichia coli 4KI-P1。
(3)采用基因工程手段在大肠杆菌Escherichia coli 4KI中敲除特异性葡萄糖转运蛋白基因ptsG,阻断磷酸转移酶系统,解除碳源代谢阻遏,增强木糖的转运及利用,构建菌株Escherichia coli 4KI01。所述葡萄糖转运蛋白基因ptsG核苷酸序列如SEQ ID NO.7所示。
(4)将实验室前期构建的pETPtac-xylBC质粒经热激转化法导入宿主Escherichiacoli 4KI01中得到工程菌株,命名为Escherichia coli 4KI01-P1。
(5)采用基因工程手段在大肠杆菌Escherichia coli 4KI01中基因组原xylA的位置敲入来源于月柄杆菌(Caulobacter crescentus)的木糖脱氢酶基因xylB与木糖酸脱水酶基因xylC,实现木糖到木糖酸的转化,构建菌株Escherichia coli 4KI02。所述木糖脱氢酶与木糖酸脱水酶基因xylBC核苷酸序列如SEQ ID NO.8所示;
(6)采用基因工程手段在大肠杆菌Escherichia coli 4KI02中敲除葡萄糖-6-磷酸异构酶编码基因pgi,增强NADPH供给,构建菌株Escherichia coli 4KI03。所述葡萄糖-6-磷酸异构酶基因pgi核苷酸序列如SEQ ID NO.9所示;
具体操作方法是:
(1)敲除方法:本发明所用基因敲除方法为一步法敲除技术(Datsenko KA etal.,Proc.Natl.Acad.Sci.USA.,2000,97:6640-6645)。该方法中,pKD4(CGSC7632)、pKD46(CGSC7669)、pCP20(CGSC14177)购自大肠杆菌基因保藏中心(纽黑文,美国,康涅狄格州)。
(2)突变片段的获得:上述方法中所用的核苷酸突变片段具有敲除基因上下游两个同源臂及卡那霉素抗性基因盒。本发明所用到的核苷酸突变片段有两种获得方式。其中xylA、ptsG、pgi基因的核苷酸突变片段通过PCR直接扩增获得。其模板购自大肠杆菌基因保藏中心(纽黑文,美国,康涅狄格州)。yagE、yjhH的基因敲除、xylD&kdcA与xylBC基因敲入的核苷酸突变片段则通过重组PCR获得。其中扩增核苷酸突变片段的引物序列如下:
核苷酸突变片段直接扩增引物
ΔxylA-F:TCACCGCGATAAACGTAACC(SEQ ID NO.10)
ΔxylA-R:CGGCAATACCCAATGCTTTA(SEQ ID NO.11)
ΔptsG-F:GTTTCACATCGACGCTTCCC(SEQ ID NO.12)
ΔptsG-R:TGCCTGTCATGCCAGAGTTG(SEQ ID NO.13)
Δpgi-F:ATATCTGGCTCTGCACGACC(SEQ ID NO.14)
Δpgi-R:CTTTAGTCGTGGCTGAACAG(SEQ ID NO.15)
核苷酸突变片段重组PCR引物
ΔyagE-F1:CTCCATAAACGGGTTCTTATGCCTT(SEQ ID NO.16)
ΔyagE-R1:CTCCAGCCTACACGAGATCTCCTTG(SEQ ID NO.17)
ΔyagE-F2:GCAAGGAGATCTCGTGTAGGCTGGA(SEQ ID NO.18)
ΔyagE-R2:GTTATCGTCCGGCATGGGAATTAGC(SEQ ID NO.19)
ΔyagE-F3:GGCTAATTCCCATGCCGGACGATAA(SEQ ID NO.20)
ΔyagE-R3:TCTGCATGCCGATCTCCCAATGCCC(SEQ ID NO.21)
ΔyihH-F1:ATACGCGCAATACATTTACCGATAAAA(SEQ ID NO.22)
ΔyihH-R1:CTCCAGCCTACACTACCTCAGTTTC(SEQ ID NO.23)
ΔyihH-F2:GGAAACTGAGGTAGTGTAGGCTGGA(SEQ ID NO.24)
ΔyihH-R2:ATGAGTTTCTCCATGGGAATTAGCC(SEQ ID NO.25)
ΔyihH-F3:GGCTAATTCCCATGGAGAAACTCATGT(SEQ ID NO.26)
ΔyihH-R3:TTCATCTGGATGTCCAGTTCGTAAT(SEQ ID NO.27)
ΔxynR::xylD&kdcA-F1:CTGGATCTGCGCCTGTTGGCCCCGA(SEQ ID NO.28)
ΔxynR::xylD&kdcA-R1:CCTAATGCAGGAGTCGCATAAATGCTGGCATGTCCACGCT(SEQ IDNO.29)
ΔxynR::xylD&kdcA-F2:AGCGTGGACATGCCAGCATTTATGCGACTCCTGCATTAGG(SEQ IDNO.30)
ΔxynR::xylD&kdcA-R2:GAAGCAGCTCCAGCCTACACCAAAAAACCCCTCAAGACCC(SEQ IDNO.31)
ΔxynR::xylD&kdcA-F3:GGGTCTTGAGGGGTTTTTTGGTGTAGGCTGGAGCTGCTTC(SEQ IDNO.32)
ΔxynR::xylD&kdcA-R3:CTACGAGCCGGTCTAACGGCATGGGAATTAGCCATGGTCC(SEQ IDNO.33)
ΔxynR::xylD&kdcA-F4:GGACCATGGCTAATTCCCATGCCGTTAGACCGGCTCGTAG(SEQ IDNO.34)
ΔxynR::xylD&kdcA-R4:CGCTTGACCCGGAGCTGCAGACCCT(SEQ ID NO.35)
ΔxylA::xylBC-F1:CGGAACAATATCGACCAGGGCTTTT(SEQ ID NO.36)
ΔxylA::xylBC-R1:TGGGATAGATGGCTGAGGACATATTGAACTCCATAATCAGGTAAT(SEQ IDNO.37)
ΔxylA::xylBC-F2:ATTACCTGATTATGGAGTTCAATATGTCCTCAGCCATCTATCCCAG(SEQID NO.38)
ΔxylA::xylBC-R2:TTCGAAGCAGCTCCAGCCTACACTTAGACAAGGCGGACCTCATGCT(SEQID NO.39)
ΔxylA::xylBC-F3:AGCATGAGGTCCGCCTTGTCTAAGTGTAGGCTGGAGCTGCTTCGAA(SEQID NO.40)
ΔxylA::xylBC-R3:CCAACGGACTGCACAGTTAGCCGATGGGAATTAGCCATGGTCCATA(SEQID NO.41)
ΔxylA::xylBC-F4:TATGGACCATGGCTAATTCCCATCGGCTAACTGTGCAGTCCGTTGG(SEQID NO.42)
ΔxylA::xylBC-R4:CAGGTAACAAAGCACCAGTAAT(SEQ ID NO.43)
(3)感受态制备与电转:将pTKRED质粒采用化学法导入目的菌株后,用添加了50μg/mL壮观霉素抗性的LB固体平板筛选重组菌株,验证后培养并进行感受态制备。用添加了50μg/mL壮观霉素抗性的50mL LB液体培养基培养重组菌株,30℃培养30分钟后加入终浓度为0.5mM IPTG诱导,待OD至0.5~0.6时冰浴10min,然后于4℃、6000rpm离心10min,收菌、去上清并用预冷的超纯水洗涤菌体两遍,用预冷的10%(v/v)甘油洗涤一遍。最后,加入200μL预冷的10%甘油重悬,制备成感受态细胞,100μL/管分装至预冷离心管,置于-80℃中冷冻保存。电转时每管感受态细胞加入10μL敲除片段,混匀后转移至2mm电转杯中进行电转。电转化条件为:1.25kV/mm,200Ω,25μF。电转后立即加入900μL LB液体培养基混匀,转移菌液至离心管,37℃孵育1h,涂布含卡那霉素的LB平板,过夜培养12h后,挑单一菌落PCR验证。
(4)卡那霉素抗性基因消除:将PCR验证正确的菌株采用化学法导入pCP20质粒,在30℃,180转摇床中孵育40分钟后涂布含有40μg/mL氯霉素抗性LB平板,30℃培养箱培养16~17小时后挑取单一菌落进行验证。将验证正确的菌株接种至无抗LB液体培养基,放置42℃,180转摇床培养2代后,37℃划线培养。挑取同一菌落分别点板至LB无抗平板、40μg/mL氯霉素抗性LB平板和50μg/mL卡那霉素抗性平板上,培养12~13小时。选择无抗平板上生长的,但在抗性平板上不生长的菌落进行菌株PCR验证。
将验证正确的菌株保菌,待用。
实施例2:Escherichia coli 4KI-P1、Escherichia coli 4KI01-P1摇瓶内发酵生产1,2,4-丁三醇
(1)平板培养:将菌株大肠埃希氏菌Escherichia coli 4KI-P1、Escherichiacoli 4KI01-P1划线到含有质量体积比为1.5~1.8%琼脂的LB平板上,37±1℃培养12±1小时;
(2)一级种子:在无菌的条件下,用无菌的牙签挑取步骤(1)平板上的一个单菌落,然后接种到5mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(3)二级种子:在无菌条件下,取步骤(2)培养的菌液以体积比为1~2%的接种量,接种到5mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(4)发酵培养:在无菌条件下,取步骤(3)所培养的菌液以体积比为2%的接种量,接种到含有50mL LB液体培养基的摇瓶中,同时添加10g/L木糖,5g/L葡萄糖及5g/L乳糖。以如下发酵培养条件培养:摇床转速为200rpm,培养温度为30℃,每2h采用10M的氢氧化钠调节pH至7.0左右,选择在木糖浓度低于5g/L时进行补料,补加10g/L木糖。培养时间为24~36小时。
上述(1)~(4)所用LB培养基配方:蛋白胨10g/L;酵母粉5g/L;NaCl 10g/L;LB培养基在121℃条件下高压灭菌20分钟。
木糖、葡萄糖和乳糖单独在115℃条件下灭菌20分钟。
其结果显示,经过代谢工程改造,敲除ptsG之后1,2,4-丁三醇产量大幅提高,Escherichia coli 4KI01-P1能够生产1,2,4-丁三醇9.8g/L,其结果如图1所示。
实施例3:利用Escherichia coli 4KI02、Escherichia coli 4KI03以木糖为底物1L发酵罐内发酵生产1,2,4-丁三醇
(1)平板培养:将菌株大肠埃希氏菌Escherichia coli 4KI02、Escherichia coli4KI03划线到含有质量体积比为1.5~1.8%琼脂的LB平板上,37±1℃培养12±1小时;
(2)一级种子:在无菌的条件下,用无菌的牙签挑取步骤(1)平板上的一个单菌落,然后接种到5mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(3)二级种子:在无菌条件下,取步骤(2)培养的菌液以体积比为1~2%的接种量,接种到100mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(4)发酵培养:在无菌条件下,取步骤(3)所培养的菌液以体积比为5%~6%的接种量,接种到含有0.8L LB液体培养基的1-L发酵罐中,同时添加30g/L木糖,10g/L葡萄糖及10g/L乳糖。以如下发酵培养条件培养:搅拌桨转速为400rpm,通气量为1.5vvm,培养温度为30℃,采用10M的氢氧化钠自动调节pH至7.0,必要时采用消泡剂消泡。发酵初始木糖浓度为30g/L,选择在木糖浓度低于10g/L时进行补料,补加至培养基中木糖浓度为30~40g/L。培养时间为36~48小时。
上述(1)~(4)所用LB培养基配方:蛋白胨10g/L;酵母粉5g/L;NaCl 10g/L;LB培养基在121℃条件下高压灭菌20分钟。
木糖、葡萄糖和乳糖单独在115℃条件下灭菌20分钟。
其结果显示,敲除pgi后,增强了NADPH水平,该菌能够生产1,2,4-丁三醇27.2g/L,提高了15.5%,生产效率为0.57g/[L·h],其结果如图1所示。
实施例4:利用Escherichia coli 4KI03以木糖为底物5L发酵罐内发酵生产1,2,4-丁三醇
(1)平板培养:将菌株大肠埃希氏菌Escherichia coli 4KI03划线到含有质量体积比为1.5~1.8%琼脂的LB平板上,37±1℃培养12±1小时;
(2)一级种子:在无菌的条件下,用无菌的牙签挑取步骤(1)平板上的一个单菌落,然后接种到5mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(3)二级种子:在无菌条件下,取步骤(2)培养的菌液以体积比为1~2%的接种量,接种到100mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(4)发酵培养:在无菌条件下,取步骤(3)所培养的菌液以体积比为5%~6%的接种量,接种到含有5L LB液体培养基的7.5-L发酵罐中,同时添加30g/L木糖,10g/L葡萄糖及10g/L乳糖。以如下发酵培养条件培养:搅拌桨转速为400rpm,通气量为1.5vvm,培养温度为30℃,采用10M的氢氧化钠自动调节pH至7.0,必要时采用消泡剂消泡。发酵初始木糖浓度为30g/L,选择在木糖浓度低于10g/L时进行补料,补加至培养基中木糖浓度为30~40g/L。培养时间为36~48小时。
上述(1)~(4)所用LB培养基配方:蛋白胨10g/L;酵母粉5g/L;NaCl 10g/L;LB培养基在121℃条件下高压灭菌20分钟。
木糖、葡萄糖和乳糖单独在115℃条件下灭菌20分钟。
其结果显示,在5L发酵罐内,该菌能够生产1,2,4-丁三醇36.63g/L,生产效率为1.14g/[L·h],其结果如图2所示。
实施例5:利用Escherichia coli 4KI03以玉米芯水解液为底物5L发酵罐内发酵生产1,2,4-丁三醇
(1)平板培养:将菌株大肠埃希氏菌Escherichia coli 4KI03划线到含有质量体积比为1.5~1.8%琼脂的LB平板上,37±1℃培养12±1小时;
(2)一级种子:在无菌的条件下,用无菌的牙签挑取步骤(1)平板上的一个单菌落,然后接种到5mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(3)二级种子:在无菌条件下,取步骤(2)培养的菌液以体积比为1~2%的接种量,接种到100mL的LB液体培养基中,37±1℃摇床振荡培养12±1小时;
(4)发酵培养:在无菌条件下,取步骤(3)所培养的菌液以体积比为5%~6%的接种量,接种到含有5L LB液体培养基的7.5-L发酵罐中,同时添加玉米芯水解液及10g/L乳糖。以如下发酵培养条件培养:搅拌桨转速为400rpm,通气量为1.5vvm,培养温度为30℃,采用10M的氢氧化钠自动调节pH至7.0,必要时采用消泡剂消泡。添加玉米芯水解液浓缩液后,发酵初始木糖浓度为30g/L,葡萄糖浓度为3g/L。选择在木糖浓度低于10g/L时进行补料,补加玉米芯水解液浓缩液至培养基中木糖浓度为30~40g/L。培养时间为36~48小时。
上述(1)~(4)所用LB培养基配方:蛋白胨10g/L;酵母粉5g/L;NaCl 10g/L;LB培养基在121℃条件下高压灭菌20分钟。
玉米芯水解液单独在115℃条件下灭菌20分钟。
玉米芯水解液成分:木糖118.5g/L;葡萄糖11.5g/L;阿拉伯糖11.8g/L;甲酸1.4g/L;乙醇0.83g/L;乙酸0.34g/L;糠醛13.5ppm等。与其相似的玉米芯水解液含木糖及葡萄糖浓度比例约为10:1。玉米芯水解液单独在115℃条件下灭菌20分钟。
其结果显示,该菌能够生产1,2,4-丁三醇43.4g/L,生产效率为1.09g/[L·h],其结果如图3所示。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 山东大学
<120> 敲除葡萄糖-6-磷酸异构酶基因的重组大肠杆菌及其在生产1,2,4-丁三醇中的应用
<130>
<160> 43
<170> PatentIn version 3.3
<210> 1
<211> 1323
<212> DNA
<213> xylA
<400> 1
atgcaagcct attttgacca gctcgatcgc gttcgttatg aaggctcaaa atcctcaaac 60
ccgttagcat tccgtcacta caatcccgac gaactggtgt tgggtaagcg tatggaagag 120
cacttgcgtt ttgccgcctg ctactggcac accttctgct ggaacggggc ggatatgttt 180
ggtgtggggg cgtttaatcg tccgtggcag cagcctggtg aggcactggc gttggcgaag 240
cgtaaagcag atgtcgcatt tgagtttttc cacaagttac atgtgccatt ttattgcttc 300
cacgatgtgg atgtttcccc tgagggcgcg tcgttaaaag agtacatcaa taattttgcg 360
caaatggttg atgtcctggc aggcaagcaa gaagagagcg gcgtgaagct gctgtgggga 420
acggccaact gctttacaaa ccctcgctac ggcgcgggtg cggcgacgaa cccagatcct 480
gaagtcttca gctgggcggc aacgcaagtt gttacagcga tggaagcaac ccataaattg 540
ggcggtgaaa actatgtcct gtggggcggt cgtgaaggtt acgaaacgct gttaaatacc 600
gacttgcgtc aggagcgtga acaactgggc cgctttatgc agatggtggt tgagcataaa 660
cataaaatcg gtttccaggg cacgttgctt atcgaaccga aaccgcaaga accgaccaaa 720
catcaatatg attacgatgc cgcgacggtc tatggcttcc tgaaacagtt tggtctggaa 780
aaagagatta aactgaacat tgaagctaac cacgcgacgc tggcaggtca ctctttccat 840
catgaaatag ccaccgccat tgcgcttggc ctgttcggtt ctgtcgacgc caaccgtggc 900
gatgcgcaac tgggctggga caccgaccag ttcccgaaca gtgtggaaga gaatgcgctg 960
gtgatgtatg aaattctcaa agcaggcggt ttcaccaccg gtggtctgaa cttcgatgcc 1020
aaagtacgtc gtcaaagtac tgataaatat gatctgtttt acggtcatat cggcgcgatg 1080
gatacgatgg cactggcgct gaaaattgca gcgcgcatga ttgaagatgg cgagctggat 1140
aaacgcatcg cgcagcgtta ttccggctgg aatagcgaat tgggccagca aatcctgaaa 1200
ggccaaatgt cactggcaga tttagccaaa tatgctcagg aacatcattt gtctccggtg 1260
catcagagtg gtcgccagga acaactggaa aatctggtaa accattatct gttcgacaaa 1320
taa 1323
<210> 2
<211> 906
<212> DNA
<213> yjhH
<400> 2
atgaaaaaat tcagcggcat tattccaccg gtatccagca cgtttcatcg tgacggaacc 60
cttgataaaa aggcaatgcg cgaagttgcc gacttcctga ttaataaagg ggtcgacggg 120
ctgttttatc tgggtaccgg tggtgaattt agccaaatga atacagccca gcgcatggca 180
ctcgccgaag aagctgtaac cattgtcgac gggcgagtgc cggtattgat tggcgtcggt 240
tccccttcca ctgacgaagc ggtcaaactg gcgcagcatg cgcaagccta cggcgctgat 300
ggtatcgtcg ccatcaaccc ctactactgg aaagtcgcac cacgaaatct tgacgactat 360
taccagcaga tcgcccgtag cgtcacccta ccggtgatcc tgtacaactt tccggatctg 420
acgggtcagg acttaacccc ggaaaccgtg acgcgtctgg ctctgcaaaa cgagaatatc 480
gttggcatca aagacaccat cgacagcgtt ggtcacttgc gtacgatgat caacacagtt 540
aagtcggtac gcccgtcgtt ttcggtattc tgcggttacg atgatcattt gctgaatacg 600
atgctgctgg gcggcgacgg tgcgataacc gccagcgcta actttgctcc ggaactctcc 660
gtcggcatct accgcgcctg gcgtgaaggc gatctggcga ccgctgcgac gctgaataaa 720
aaactactac aactgcccgc tatttacgcc ctcgaaacac cgtttgtctc actgatcaaa 780
tacagcatgc agtgtgtcgg gctgcctgta gagacatatt gcttaccacc gattcttgaa 840
gcatctgaag aagcaaaaga taaagtccac gtgctgctta ccgcgcaggg cattttacca 900
gtctga 906
<210> 3
<211> 909
<212> DNA
<213> yagE
<400> 3
atgccgcagt ccgcgttgtt cacgggaatc attccccctg tctccaccat ttttaccgcc 60
gacggccagc tcgataagcc gggcaccgcc gcgctgatcg acgatctgat caaagcaggc 120
gttgacggcc tgttcttcct gggcagcggt ggcgagttct cccagctcgg cgccgaagag 180
cgtaaagcca ttgcccgctt tgctatcgat catgtcgatc gtcgcgtgcc ggtgctgatc 240
ggcaccggcg gcaccaacgc ccgggaaacc atcgaactca gccagcacgc gcagcaggcg 300
ggcgcggacg gcatcgtggt gatcaacccc tactactgga aagtgtcgga agcgaacctg 360
atccgctatt tcgagcaggt ggccgacagc gtcacgctgc cggtgatgct ctataacttc 420
ccggcgctga ccgggcagga tctgactccg gcgctggtga aaaccctcgc cgactcgcgc 480
agcaatatta tcggcatcaa agacaccatc gactccgtcg cccacctgcg cagcatgatc 540
cataccgtca aaggtgccca tccgcacttc accgtgctct gcggctacga cgatcatctg 600
ttcaataccc tgctgctcgg cggcgacggg gcgatatcgg cgagcggcaa ctttgccccg 660
caggtgtcgg tgaatcttct gaaagcctgg cgcgacgggg acgtggcgaa agcggccggg 720
tatcatcaga ccttgctgca aattccgcag atgtatcagc tggatacgcc gtttgtgaac 780
gtgattaaag aggcgatcgt gctctgcggt cgtcctgtct ccacgcacgt gctgccgccc 840
gcctcgccgc tggacgagcc gcgcaaggcg cagctgaaaa ccctgctgca acagctcaag 900
ctttgctga 909
<210> 4
<211> 759
<212> DNA
<213> xynR
<400> 4
atgccgatta ttcagtctgt tgaacgtgcg ttgcagatcc tcgacctgtt caacgagcag 60
gccaccgagc ttaagatcac cgacatcagc aaactgatgg ggctgagcaa gagtaccctc 120
cactcgctgc taaaaaccct gcagcttcac ggctatatcg atcagaaccc ggagaacggc 180
aagtatcgcc tcggcatgaa gctggtcgag cgcggccatt ttgtcgtggg ctccatcgat 240
attcggcaga aggcaaaagg ctggctgacg gagctgtccc ggcggaccgg gcagaccacc 300
catctgggga tcctggacgg gcgtgaaggg gtctatatcg agaagattga aggcaagctg 360
gccgccatcg cctattcacg catcggccgc cgcctgccgg tgcacgccac cgccatcggc 420
aaggtgttga ttgcctggct gggcgaggcc gagctgaacg ccctgctgga gggctatcag 480
tacactacct ttacgcccgc caccctcgcg tctcgcgaag ccttaatgag cgccctggcg 540
cagacccgcg agcaaggcta cgccctggac agcgaagaga acgagcaggg cgtgcgctgc 600
gtggcggtgc cggtgtggaa ccacgagtcc cgcgtcatcg ccgccctgag cctgtcgacg 660
ctgacctccc gcgtggacga cgcggagctg gctaatttcc gcgagcagct tcagcaggcc 720
gggctcgcgc tctcgcgcgc gctgggctac ccggcctga 759
<210> 5
<211> 1788
<212> DNA
<213> xylD
<400> 5
atgcgtagtg ccctgagtaa tcgtaccccg cgccgttttc gtagccgcga ttggtttgat 60
aatccggatc atattgatat gaccgcactg tatctggaac gctttatgaa ttatggcatt 120
accccggaag aactgcgtag tggtaaaccg attattggca ttgcccagac cggtagtgat 180
attagtccgt gtaatcgcat tcatctggat ctggtgcagc gtgttcgcga tggcattcgc 240
gatgccggtg gcattccgat ggaatttccg gttcatccga tttttgaaaa ttgccgtcgt 300
ccgaccgccg cactggatcg caatctgagc tatctgggcc tggttgaaac cctgcatggt 360
tatccgattg atgcagttgt tctgaccacc ggctgcgata aaaccacccc ggccggtatt 420
atggcagcaa ccaccgtgaa tattccggcc attgttctga gcggcggtcc gatgctggat 480
ggttggcatg aaaatgaact ggtgggcagc ggcaccgtta tttggcgcag tcgtcgcaaa 540
ctggccgcag gcgaaattac cgaagaagag tttattgatc gtgcagcaag tagtgcaccg 600
agcgccggcc attgtaatac catgggtaca gcaagcacca tgaatgcagt ggccgaagca 660
ctgggcctga gtctgaccgg ctgcgccgct attccggccc cttatcgtga acgtggccag 720
atggcatata aaaccggcca gcgcattgtt gatctggcat atgatgatgt gaaaccgctg 780
gatattctga ccaaacaggc atttgaaaat gccattgcac tggttgcagc cgccggtggc 840
agcaccaatg cacagccgca tattgttgcc atggcccgtc atgccggcgt ggaaattacc 900
gcagatgatt ggcgcgccgc atatgatatt ccgctgattg tgaatatgca gccggcaggc 960
aaatatctgg gtgaacgttt tcatcgcgca ggtggtgccc cggcagtgct gtgggaactg 1020
ctgcagcagg gtcgcctgca tggcgatgtt ctgaccgtga ccggcaaaac catgagtgaa 1080
aatctgcagg gccgcgaaac cagcgatcgc gaagttattt ttccgtatca tgaaccgctg 1140
gccgaaaaag ccggttttct ggttctgaaa ggcaatctgt ttgattttgc aattatgaaa 1200
agcagtgtga ttggtgaaga atttcgtaaa cgctatctga gtcagccggg tcaggaaggt 1260
gtgtttgaag cccgtgccat tgtttttgat ggcagcgatg attatcataa acgtattaat 1320
gacccggccc tggaaattga tgaacgttgc attctggtta ttcgcggtgc cggcccgatt 1380
ggctggccgg gtagtgcaga agtggtgaat atgcaaccgc cggatcatct gctgaaaaaa 1440
ggcattatga gcctgccgac cctgggtgac ggtcgccaga gcggtacagc agatagtccg 1500
agcattctga atgccagccc ggaaagcgcc attggtggcg gcctgagttg gctgcgcacc 1560
ggtgacacca ttcgcattga tctgaatacc ggccgctgcg atgccctggt tgatgaagca 1620
accattgcag cccgtaaaca ggatggtatt ccggcagttc cggccaccat gaccccgtgg 1680
caggaaatct atcgtgcaca tgccagccag ctggataccg gtggtgttct ggaatttgca 1740
gtgaaatatc aggatctggc cgcaaaactg ccgcgccata atcattaa 1788
<210> 6
<211> 1644
<212> DNA
<213> kdcA
<400> 6
atgtacaccg ttggcgatta tctgctggat cgtctgcatg aactgggtat tgaagaaatt 60
tttggtgttc cgggtgacta taatctgcag tttctggatc agattattag tcgcgaagat 120
atgaaatgga ttggtaatgc caatgaactg aatgcaagtt atatggccga tggttatgcc 180
cgcaccaaaa aagcagcagc ctttctgacc acctttggcg tgggtgaact gagtgcaatt 240
aatggtctgg ccggtagtta tgccgaaaat ctgccggtgg ttgaaattgt tggcagtccg 300
accagcaaag ttcagaatga tggtaaattt gtgcatcata ccctggcaga tggcgatttt 360
aaacatttta tgaaaatgca cgagccggtg accgcagccc gtaccctgct gaccgcagaa 420
aatgcaacct atgaaattga tcgtgtgctg agtcagctgc tgaaagaacg caaaccggtt 480
tatattaatc tgccggttga tgttgccgcc gccaaagcag aaaaaccggc cctgagtctg 540
gaaaaagaaa gcagcaccac caataccacc gaacaggtta ttctgagcaa aattgaagaa 600
agcctgaaaa atgcacagaa accggttgtt attgcaggcc atgaagtgat tagctttggt 660
ctggaaaaaa ccgtgaccca gtttgttagc gaaaccaaac tgccgattac caccctgaat 720
tttggtaaaa gtgcagtgga tgaaagcctg ccgagttttc tgggcatcta taatggcaaa 780
ctgagtgaaa ttagtctgaa aaatttcgtg gaaagcgcag attttattct gatgctgggt 840
gttaaactga ccgatagcag caccggcgcc tttacccatc atctggatga aaataagatg 900
attagcctga atatcgatga aggtattatt tttaacaagg tggttgaaga tttcgatttt 960
cgtgcagtgg tgagtagtct gagtgaactg aaaggcattg aatatgaagg tcagtatatt 1020
gataagcagt atgaagagtt tattccgagt agcgccccgc tgagtcagga tcgcctgtgg 1080
caggccgttg aaagtctgac ccagagtaat gaaaccattg ttgccgaaca gggtaccagc 1140
tttttcggcg caagtaccat ttttctgaaa agtaatagcc gctttatcgg ccagccgctg 1200
tggggtagta ttggttatac ctttccggca gccctgggca gccagattgc agataaagaa 1260
agccgtcatc tgctgtttat tggcgatggt agtctgcagc tgaccgttca ggaactgggt 1320
ctgagcattc gtgaaaaact gaatccgatt tgttttatta tcaacaacga cggctatacc 1380
gtggaacgtg aaattcatgg tccgacccag agttataatg atattccgat gtggaattac 1440
agcaaactgc cggaaacctt tggcgcaacc gaagatcgtg ttgttagtaa aattgtgcgt 1500
accgaaaatg aatttgttag cgtgatgaaa gaagcacagg ccgatgttaa tcgtatgtat 1560
tggattgaac tggtgctgga aaaagaggat gcaccgaaac tgctgaaaaa gatgggcaaa 1620
ctgtttgccg aacagaataa gtaa 1644
<210> 7
<211> 1434
<212> DNA
<213> ptsG
<400> 7
atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcgctgat gctgccggta 60
tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120
cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180
ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240
gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300
ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360
gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420
cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480
gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540
accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600
atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660
cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720
atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780
ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840
ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900
gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960
ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020
ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080
ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140
ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200
atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260
tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320
aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380
actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434
<210> 8
<211> 1741
<212> DNA
<213> xylBC
<400> 8
atgtcctcag ccatctatcc cagcctgaag ggcaagcgcg tcgtcatcac cggcggcggc 60
tcgggcatcg gggccggcct caccgccggc ttcgcccgtc agggcgcgga ggtgatcttc 120
ctcgacatcg ccgacgagga ctccagggct cttgaggccg agctggccgg ctcgccgatc 180
ccgccggtct acaagcgctg cgacctgatg aacctcgagg cgatcaaggc ggtcttcgcc 240
gagatcggcg acgtcgacgt gctggtcaac aacgccggca atgacgaccg ccacaagctg 300
gccgacgtga ccggcgccta ttgggacgag cggatcaacg tcaacctgcg ccacatgctg 360
ttctgcaccc aggccgtcgc gccgggcatg aagaagcgtg gcggcggggc ggtgatcaac 420
ttcggttcga tcagctggca cctggggctt gaggacctcg tcctctacga aaccgccaag 480
gccggcatcg aaggcatgac ccgcgcgctg gcccgggagc tgggtcccga cgacatccgc 540
gtcacctgcg tggtgccggg caacgtcaag accaagcgcc aggagaagtg gtacacgccc 600
gaaggcgagg cccagatcgt ggcggcccaa tgcctgaagg gccgcatcgt cccggagaac 660
gtcgccgcgc tggtgctgtt cctggcctcg gatgacgcgt cgctctgcac cggccacgaa 720
tactggatcg acgccggctg gcgttgacct aagaaaactg tcatcccggc ccagcgtgaa 780
gcgcgccgag ccgggaccac ggcaagcgcc acgcgtccgg aggtcccggc tctccgctgt 840
gctacggccg ggatgacaga ggaatgattg tatgaccgct caagtcactt gcgtatggga 900
tctgaaggcc acgttgggcg aaggcccgat ctggcatggc gacaccctgt ggttcgtcga 960
catcaagcag cgtaaaatcc acaactacca ccccgccacc ggcgagcgct tcagcttcga 1020
cgcgccggat caggtgacct tcctcgcgcc gatcgtcggc gcgaccggct ttgtcgtcgg 1080
tctgaagacc gggattcacc gcttccaccc ggccacgggc ttcagcctgc tgctcgaggt 1140
cgaggacgcg gcgctgaaca accgccccaa cgacgccacg gtcgacgcgc aaggccgtct 1200
gtggttcggc accatgcacg acggggaaga gaacaatagc ggctcgctct atcggatgga 1260
cctcaccggc gtcgcccgga tggaccgcga catctgcatc accaacggcc cgtgcgtctc 1320
gcccgacggc aagaccttct accacaccga caccctggaa aagacgatct acgccttcga 1380
cctggccgag gacggcctgc tgtcgaacaa gcgcgtcttc gtgcagttcg ccctgggcga 1440
cgatgtctat ccggacggtt cggtcgtcga ttccgaaggc tatctgtgga ccgccctgtg 1500
gggcggtttc ggcgcggtcc gcttctcgcc gcaaggcgac gccgtgacgc gcatcgaact 1560
gcccgccccc aacgtcacca agccctgctt cggcgggcct gacctgaaga ccctctattt 1620
caccaccgcc cgcaagggcc tgagcgacga gaccctggcc cagtacccgc tggccggcgg 1680
tgtgttcgcc gttccggtcg atgtggccgg ccaaccccag catgaggtcc gccttgtcta 1740
a 1741
<210> 9
<211> 1650
<212> DNA
<213> pgi
<400> 9
atgaaaaaca tcaatccaac gcagaccgct gcctggcagg cactacagaa acacttcgat 60
gaaatgaaag acgttacgat cgccgatctt tttgctaaag acggcgatcg tttttctaag 120
ttctccgcaa ccttcgacga tcagatgctg gtggattact ccaaaaaccg catcactgaa 180
gagacgctgg cgaaattaca ggatctggcg aaagagtgcg atctggcggg cgcgattaag 240
tcgatgttct ctggcgagaa gatcaaccgc actgaaaacc gcgccgtgct gcacgtagcg 300
ctgcgtaacc gtagcaatac cccgattttg gttgatggca aagacgtaat gccggaagtc 360
aacgcggtgc tggagaagat gaaaaccttc tcagaagcga ttatttccgg tgagtggaaa 420
ggttataccg gcaaagcaat cactgacgta gtgaacatcg ggatcggcgg ttctgacctc 480
ggcccataca tggtgaccga agctctgcgt ccgtacaaaa accacctgaa catgcacttt 540
gtttctaacg tcgatgggac tcacatcgcg gaagtgctga aaaaagtaaa cccggaaacc 600
acgctgttct tggtagcatc taaaaccttc accactcagg aaactatgac caacgcccat 660
agcgcgcgtg actggttcct gaaagcggca ggtgatgaaa aacacgttgc aaaacacttt 720
gcggcgcttt ccaccaatgc caaagccgtt ggcgagtttg gtattgatac tgccaacatg 780
ttcgagttct gggactgggt tggcggccgt tactctttgt ggtcagcgat tggcctgtcg 840
attgttctct ccatcggctt tgataacttc gttgaactgc tttccggcgc acacgcgatg 900
gacaagcatt tctccaccac gcctgccgag aaaaacctgc ctgtactgct ggcgctgatt 960
ggcatctggt acaacaattt ctttggtgcg gaaactgaag cgattctgcc gtatgaccag 1020
tatatgcacc gtttcgcggc gtacttccag cagggcaata tggagtccaa cggtaagtat 1080
gttgaccgta acggtaacgt tgtggattac cagactggcc cgattatctg gggtgaacca 1140
ggcactaacg gtcagcacgc gttctaccag ctgatccacc agggaaccaa aatggtaccg 1200
tgcgatttca tcgctccggc tatcacccat aacccgctct ctgatcatca ccagaaactg 1260
ctgtctaact tcttcgccca gaccgaagcg ctggcgtttg gtaaatcccg cgaagtggtt 1320
gagcaggaat atcgtgatca gggtaaagat ccggcaacgc ttgactacgt ggtgccgttc 1380
aaagtattcg aaggtaaccg cccgaccaac tccatcctgc tgcgtgaaat cactccgttc 1440
agcctgggtg cgttgattgc gctgtatgag cacaaaatct ttactcaggg cgtgatcctg 1500
aacatcttca ccttcgacca gtggggcgtg gaactgggta aacagctggc gaaccgtatt 1560
ctgccagagc tgaaagatga taaagaaatc agcagccacg atagctcgac caatggtctg 1620
attaaccgct ataaagcgtg gcgcggttaa 1650
<210> 10
<211> 20
<212> DNA
<213> 人工序列
<400> 10
tcaccgcgat aaacgtaacc 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<400> 11
cggcaatacc caatgcttta 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<400> 12
gtttcacatc gacgcttccc 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<400> 13
tgcctgtcat gccagagttg 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列
<400> 14
atatctggct ctgcacgacc 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列
<400> 15
ctttagtcgt ggctgaacag 20
<210> 16
<211> 25
<212> DNA
<213> 人工序列
<400> 16
ctccataaac gggttcttat gcctt 25
<210> 17
<211> 25
<212> DNA
<213> 人工序列
<400> 17
ctccagccta cacgagatct ccttg 25
<210> 18
<211> 25
<212> DNA
<213> 人工序列
<400> 18
gcaaggagat ctcgtgtagg ctgga 25
<210> 19
<211> 25
<212> DNA
<213> 人工序列
<400> 19
gttatcgtcc ggcatgggaa ttagc 25
<210> 20
<211> 25
<212> DNA
<213> 人工序列
<400> 20
ggctaattcc catgccggac gataa 25
<210> 21
<211> 25
<212> DNA
<213> 人工序列
<400> 21
tctgcatgcc gatctcccaa tgccc 25
<210> 22
<211> 27
<212> DNA
<213> 人工序列
<400> 22
atacgcgcaa tacatttacc gataaaa 27
<210> 23
<211> 25
<212> DNA
<213> 人工序列
<400> 23
ctccagccta cactacctca gtttc 25
<210> 24
<211> 25
<212> DNA
<213> 人工序列
<400> 24
ggaaactgag gtagtgtagg ctgga 25
<210> 25
<211> 25
<212> DNA
<213> 人工序列
<400> 25
atgagtttct ccatgggaat tagcc 25
<210> 26
<211> 27
<212> DNA
<213> 人工序列
<400> 26
ggctaattcc catggagaaa ctcatgt 27
<210> 27
<211> 25
<212> DNA
<213> 人工序列
<400> 27
ttcatctgga tgtccagttc gtaat 25
<210> 28
<211> 25
<212> DNA
<213> 人工序列
<400> 28
ctggatctgc gcctgttggc cccga 25
<210> 29
<211> 40
<212> DNA
<213> 人工序列
<400> 29
cctaatgcag gagtcgcata aatgctggca tgtccacgct 40
<210> 30
<211> 40
<212> DNA
<213> 人工序列
<400> 30
agcgtggaca tgccagcatt tatgcgactc ctgcattagg 40
<210> 31
<211> 40
<212> DNA
<213> 人工序列
<400> 31
gaagcagctc cagcctacac caaaaaaccc ctcaagaccc 40
<210> 32
<211> 40
<212> DNA
<213> 人工序列
<400> 32
gggtcttgag gggttttttg gtgtaggctg gagctgcttc 40
<210> 33
<211> 40
<212> DNA
<213> 人工序列
<400> 33
ctacgagccg gtctaacggc atgggaatta gccatggtcc 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列
<400> 34
ggaccatggc taattcccat gccgttagac cggctcgtag 40
<210> 35
<211> 25
<212> DNA
<213> 人工序列
<400> 35
cgcttgaccc ggagctgcag accct 25
<210> 36
<211> 25
<212> DNA
<213> 人工序列
<400> 36
cggaacaata tcgaccaggg ctttt 25
<210> 37
<211> 45
<212> DNA
<213> 人工序列
<400> 37
tgggatagat ggctgaggac atattgaact ccataatcag gtaat 45
<210> 38
<211> 46
<212> DNA
<213> 人工序列
<400> 38
attacctgat tatggagttc aatatgtcct cagccatcta tcccag 46
<210> 39
<211> 46
<212> DNA
<213> 人工序列
<400> 39
ttcgaagcag ctccagccta cacttagaca aggcggacct catgct 46
<210> 40
<211> 46
<212> DNA
<213> 人工序列
<400> 40
agcatgaggt ccgccttgtc taagtgtagg ctggagctgc ttcgaa 46
<210> 41
<211> 46
<212> DNA
<213> 人工序列
<400> 41
ccaacggact gcacagttag ccgatgggaa ttagccatgg tccata 46
<210> 42
<211> 46
<212> DNA
<213> 人工序列
<400> 42
tatggaccat ggctaattcc catcggctaa ctgtgcagtc cgttgg 46
<210> 43
<211> 22
<212> DNA
<213> 人工序列
<400> 43
caggtaacaa agcaccagta at 22
Claims (11)
1.一株重组大肠杆菌,其特征在于,所述重组大肠杆菌是以E. coli W3110 (DE3)为出发菌株,通过敲除木糖异构酶xylA、2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE、木糖酸操纵子转录抑制子基因xynR、特异性葡萄糖转运蛋白基因ptsG和葡萄糖-6-磷酸异构酶编码基因pgi;同时在基因组xynR位点敲入木糖酸脱水酶基因xylD和2-酮酸脱羧酶基因kdcA,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC获得;
所述木糖异构酶xylA的核苷酸序列如SEQ ID NO.1所示;
所述2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE的核苷酸序列如SEQ ID NO.2和SEQID NO.3所示;
所述木糖酸操纵子转录抑制子基因xynR的核苷酸序列如SEQ ID NO.4所示;
所述木糖酸脱水酶基因xylD来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.5所示;2-酮酸脱羧酶基因kdcA来源于乳酸乳球菌(Lactococcus lactis),其核苷酸序列如SEQ ID NO.6所示;
所述特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi的核苷酸序列如SEQ ID NO.7和SEQ ID NO.9所示;
所述木糖脱氢酶及木糖酸内酯酶编码基因xylBC来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.8所示。
2.权利要求1所述重组大肠杆菌的构建方法,其特征在于,所述构建方法包括:敲除出发菌株E. coli W3110 (DE3)的木糖异构酶xylA;敲除2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE;敲除木糖酸操纵子转录抑制子基因xynR;同时将木糖酸脱水酶基因xylD和2-酮酸脱羧酶基因kdcA敲入基因组原xynR位点,敲除其特异性葡萄糖转运蛋白基因ptsG,并在基因组xylA基因位点敲入木糖脱氢酶及木糖酸内酯酶编码基因xylBC,以及敲除葡萄糖-6-磷酸异构酶编码基因pgi;
所述木糖异构酶xylA的核苷酸序列如SEQ ID NO.1所示;
所述2-酮基-3-脱氧木糖酸醛缩酶基因yjhH和yagE的核苷酸序列如SEQ ID NO.2和SEQID NO.3所示;
所述木糖酸操纵子转录抑制子基因xynR的核苷酸序列如SEQ ID NO.4所示;
所述木糖酸脱水酶基因xylD来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.5所示;2-酮酸脱羧酶基因kdcA来源于乳酸乳球菌(Lactococcus lactis),其核苷酸序列如SEQ ID NO.6所示;
所述特异性葡萄糖转运蛋白基因ptsG以及葡萄糖-6-磷酸异构酶编码基因pgi的核苷酸序列如SEQ ID NO.7和SEQ ID NO.9所示;
所述木糖脱氢酶及木糖酸内酯酶编码基因xylBC来源于月柄杆菌(Caulobacter crescentus),其核苷酸序列如SEQ ID NO.8所示。
3.如权利要求2所述的构建方法,其特征在于,基因敲除方法采用一步法敲除技术,所用的核苷酸突变片段具有敲除基因上下游两个同源臂及卡那霉素抗性基因盒。
4.如权利要求3所述的构建方法,其特征在于,所述xylA、ptsG和pgi基因的核苷酸突变片段通过PCR直接扩增获得;所述yagE、yjhH的基因敲除,xylD、kdcA和xylBC基因敲入的核苷酸突变片段则通过重组PCR获得。
5.如权利要求4所述的构建方法,其特征在于,其中扩增核苷酸突变片段的引物序列如SEQ ID NO.10-43所示。
6.一种菌剂,其特征在于,所述菌剂含有权利要求1所述重组大肠杆菌。
7.权利要求1所述重组大肠杆菌或权利要求6所述菌剂在以木糖或玉米芯水解液为底物发酵生产1,2,4-丁三醇中的应用。
8.一种发酵生产1,2,4-丁三醇的方法,其特征在于,所述方法包括:以木糖或玉米芯水解液为底物,生物发酵权利要求1所述重组大肠杆菌,由发酵液得到1,2,4-丁三醇。
9.如权利要求8所述方法,其特征在于,所述发酵条件是:培养温度为30±1 ℃,培养方式为搅拌培养,搅拌转速为400±50转/分钟,通气量为1.5±0.1 vvm,调节pH至7.0±0.4,培养时间为36~48小时;
以木糖为底物时,木糖与葡萄糖的含量比例为2-6:1,木糖含量不低于30g/L;不使用IPTG诱导,使用乳糖进行诱导;
以玉米芯水解液为底物时,玉米芯水解液成分中木糖与葡萄糖的含量比例在6:1~12:1,木糖含量不低于30g/L;使用乳糖进行诱导。
10.如权利要求9所述方法,其特征在于,以木糖为底物时,所述木糖与葡萄糖的含量比例为3:1,乳糖诱导使用浓度为10 g/L。
11.如权利要求9所述方法,其特征在于,以玉米芯水解液为底物时,玉米芯水解液成分中木糖与葡萄糖的含量比例10:1,乳糖诱导使用浓度为10 g/L。
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