CN116926102A - 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 - Google Patents
抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 Download PDFInfo
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Abstract
本发明公开了抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5‑ALA的方法,所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。实验证明,谷氨酸棒杆菌中全局转录调控因子基因glxR表达的抑制使得谷氨酸棒杆菌工程菌株中5‑氨基乙酰丙酸产量比CGS15‑h3提升约1.37倍。
Description
技术领域
本发明属于生物工程技术与应用领域,具体地涉及抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法。
背景技术
5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA),是生物体内合成维生素B12、血红素、叶绿素等四吡咯类化合物的重要前体,广泛存在于微生物和动植物细胞中。由于其绿色无毒、易降解且无残留的特性,作为光动力学药物、生物可降解除草剂、植物生长调节剂以及饲料添加剂等在医药以及农业等领域得到广泛应用。目前5-ALA的大规模生产主要以化学法为主,但是合成过程中存在的高成本、高污染和低得率等问题大大限制了其大规模生产与应用。鉴于此,微生物合成法应运而生,并随着合成生物学,特别是代谢工程手段的兴起,5-ALA的生物法合成得到了进一步发展。但是,大多数生物合成方法产量低,并不能达到工业化生产的要求,难以实现大规模应用。
随着基因组技术的发展,转录因子(Transfer Factor,TF)作为改变复杂微生物代谢途径的调节因子,在转录水平的基因调控方面具有独特的优势,弥补了单个关键基因作用的不足,并降低了代谢工程操作中多个关键基因组成性致死表达的可能性。尤其是全局调节因子的修饰可以在转录水平上同时干扰数百个基因的表达。基于转录因子的生物传感器具有高灵敏度和特异性,在代谢工程中发挥着重要作用,显著提高了目标化合物的产量和宿主细胞耐受性。
但是,目前尚未有抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法的报道。
发明内容
本发明的目的是克服现有技术的不足,提供抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法。
本发明的技术方案概述如下:
抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法,所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
优选地,上述方法,包括如下步骤:
1)pCRISPRi-low-GlxR质粒构建:
以pdCas9gRNA为模板,以l-GlxR-F1和cas-R1为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段1;
以pdCas9gRNA为模板,以ori-F和CRISPRi-R2为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段2;
将所述片段1和片段2连接起来,通过菌落PCR的方式进行验证得到pCRISPRi-low-GlxR质粒;
所述l-GlxR-F1的核苷酸序列如SEQ ID NO.8所示;
所述cas-R1的核苷酸序列如SEQ ID NO.3所示;
所述ori-F的核苷酸序列如SEQ ID NO.4所示;
所述CRISPRi-R2的核苷酸序列如SEQ ID NO.7所示;
2)5-氨基乙酰丙酸生产菌株构建及发酵:
将pCRISPRi-low-GlxR质粒通过电转导入谷氨酸棒杆菌CGS15-h3中,用菌落PCR的方式进行验证得到抑制全局转录调控因子基因glxR的表达菌株CGS15-l-GlxR;发酵,得到5-氨基乙酰丙酸。
本发明的优点:
实验证明,谷氨酸棒杆菌中全局转录调控因子基因glxR表达的抑制使得谷氨酸棒杆菌工程菌株中5-氨基乙酰丙酸产量比CGS15-h3提升约1.37倍。
附图说明
图1为pCRISPRi-high-GlxR质粒图谱。
图2为pCRISPRi-middle-GlxR质粒图谱。
图3为pCRISPRi-low-GlxR质粒图谱。
图4为菌株CGS15-h-GlxR,CGS15-m-GlxR和CGS15-l-GlxR在摇瓶条件下5-ALA产量图。
具体实施方式
下面结合具体实施例对本发明做进一步说明,下述实施例是为了使本领域的技术人员能够更好地理解本发明,但对本发明不作任何限制。
本发明所用到的底盘菌株谷氨酸棒杆菌Corynebacterium glutamicum ATCC13032来源为ATCC(The Global Bioresource Center,http://www.atcc.org/),购买于2012年10月。
CGS15-h3的基因型为ATCC13032△ldhA△pqo::Psod*hemA△cat::Psod*hemA△pta△ackAPsod pyc Psod ppc,△aceA::Psod*hemA,Psod tal Psod tkt,Ptuf araE(del21bp),ackA-pta::Psod xylAB:C131T,NCgl2538:C311T
pdCas9gRNA的制备参见参考文献1。
5-氨基乙酰丙酸标准品从sigma公司(http://www.sigmaaldrich.com/sigmaaldrich)购买。
所用限制性内切酶、DNA连接酶等分子生物学试剂从Thermo公司购买(http://www.thermoscientificbio.com/fermentas),所用其他生化试剂从生工生物工程(上海)股份有限公司购买(http://www.sangon.com/)。
E.coli DH5α感受态细胞、E.coli BL21感受态细胞通过常规CaCl2法制备;
LB液体培养基:酵母浸粉5g/L,胰蛋白胨10g/L,NaCl 10g/L,LB固体培养基中添加2%的琼脂粉。
CGIII培养基:酵母浸粉10g/L,胰蛋白胨10g/L,MOPS 21g/L,NaCl 2.5g/L,用5M的NaOH水溶液调节pH=7。
BHIS液体培养基:脑心浸液肉汤粉74g/L。BHIS固体培养基中添加2%的琼脂粉。
抗生素浓度为:卡那霉素40μg/mL、15μg/mL,氯霉素10μg/mL。
5-ALA的检测方法:取250μL的5-氨基乙酰丙酸标准品溶液(终浓度为1、2、4、6、8mg/L)或稀释的发酵液或酶活力测定反应终止后的反应液,加入125μL pH=4.6的乙酸钠缓冲液,然后加入62.5μL的乙酰丙酮,100℃金属浴温浴15min,冷却至室温后加入440μL现配的Modified Ehrlich's reagent试剂(0.2g对二甲氨基苯甲醛,1mL冰乙酸,1mL高氯酸,冰乙酸定容到10mL)混匀,室温反应20min后测反应液在554nm波长下的吸光值。利用测定5-氨基乙酰丙酸标准品得到的标准曲线计算发酵液或者酶活力测定反应终止后的反应液中的5-ALA含量。
实施例1
抑制谷氨酸棒杆菌(CGS15-h3)中全局转录调控因子基因glxR的表达生产5-氨基乙酰丙酸的方法,所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
实施例2
pCRISPRi-GlxR抑制载体构建
(1)GlxR表达高抑制载体pCRISPRi-high-GlxR的构建
以pdCas9gRNA为模板,以GlxR-F1(SEQ ID NO.2)、cas-R1(SEQ ID NO.3)为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段3;
以pdCas9gRNA为模板,以ori-F(SEQ ID NO.4)、GlxR-R2(SEQ ID NO.5)为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段4;
用CPEC技术将片段3和片段4连接起来,测序验证正确后通过电转化的方式转入CGS15-h3中,并通过菌落PCR的方式进行验证,得到高抑制调控因子GlxR的工程菌株,最终的pCRISPRi-high-GlxR质粒图谱如图1所示。
(2)GlxR表达中抑制载体pCRISPRi-middle-GlxR的构建
以pdCas9gRNA为模板,以m-GlxR-F1(SEQ ID NO.6)、cas-R1(SEQ ID NO.3)为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段5;
以pdCas9gRNA为模板,以ori-F(SEQ ID NO.4)、CRISPRi-R2(SEQ ID NO.7)作为上下游引物,使用Phanta DNA聚合酶进行扩增,得到线性化载体片段2;
使用CPEC技术将片段5和片段2连接起来,测序验证正确后通过电转化的方式转入CGS15-h3中,并通过菌落PCR的方式进行验证,得到中抑制调控因子GlxR的工程菌株。最终的pCRISPRi-middle-GlxR质粒图谱如图2所示。
(3)GlxR表达低抑制载体pCRISPRi-low-GlxR的构建
以pdCas9gRNA为模板,以l-GlxR-F1(SEQ ID NO.8)、cas-R1(SEQ ID NO.3)为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段1;
以pdCas9gRNA为模板,以ori-F(SEQ ID NO.4)和CRISPRi-R2(SEQ ID NO.7)作为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段2。
使用CPEC技术将片段1和片段2连接起来,测序验证正确后通过电转化的方式转入CGS15-h3中,并通过菌落PCR的方式进行验证,得到低抑制调控因子GlxR的工程菌株。最终的pCRISPRi-low-GlxR质粒图谱如图3所示。
PCR扩增体系:模板2μL,上下游引物各2μL,dNTP 1μL,phanta Buffer 25μL,灭菌的双蒸水17μL,DNA聚合酶1μL,总体积50μL。
PCR反应条件为:95℃3min,30个循环(95℃20s,58℃20s,72℃1min),72℃10min,4℃10min。采用胶回收试剂盒对PCR产物进行回收和纯化。
表1菌株构建所用引物序列
实施例2:5-ALA生产菌株构建及其摇瓶发酵
(1)5-ALA生产菌株构建
将测序结果正确的质粒pCRISPRi-high-GlxR,pCRISPRi-middle-GlxR和pCRISPRi-low-GlxR通过电转导入谷氨酸棒杆菌CGS15-h3中,均匀涂在氯霉素抗性(终浓度10μg/mL)的BHIS固体平板上。分别挑取单菌落以如下引物test-pX-1(SEQ ID NO.9)/test-pX-2(SEQ ID NO.10)进行PCR验证。测序无误的即为成功插入质粒的,能够不同程度抑制全局转录调控因子基因glxR的表达菌株的5-ALA生产菌株CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR。
(2)生产菌株的摇瓶发酵
对菌株CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR进行摇瓶发酵。
接种方式:首先将CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR在BHIS固体培养基上进行划线,置于30℃培养箱中培养约18h。挑取平板上的单菌落接种至5mL的BHIS液体培养基中,30℃,220rpm培养约12h,取1mL转接至CGIII液体培养基继续培养12h。以初始OD600约为0.5、转接葡萄糖的终浓度为10g/L的CGIII液体培养基,置于30℃,220rmp的恒温摇床中振荡培养4h至OD600约为5.0左右时,添加7.5g/L的前体物质甘氨酸。在培养期间测定其5-ALA产量(见图4)。培养72h后,菌株CGS15-h-GlxR的5-ALA产量比CGS15-h3提升约0.35倍,菌株CGS15-m-GlxR的5-ALA产量比CGS15-h3提升约0.5倍,菌株CGS15-l-GlxR的5-ALA产量为4.62g/L比CGS15-h3提升约1.37倍。
参考文献
1.Liu J.,Liu M.,Shi T.,Sun G.,Gao N.,Zhao X.,Guo X.,Ni X.,Yuan Q.,Feng J.,Liu Z.,Guo Y.,Chen J.,Wang Y.,Zheng P.,Sun J.CRISPR-assisted rationalflux-tuning and arrayed CRISPRi screening of an L-proline exporterfor L-proline hyperproduction.Nat Commun2022;13:891.
Claims (2)
1.抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法,其特征是所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法,其特征是包括如下步骤:
1)pCRISPRi-low-GlxR质粒构建:
以pdCas9gRNA为模板,以l-GlxR-F1和cas-R1为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段1;
以pdCas9gRNA为模板,以ori-F和CRISPRi-R2为上下游引物,用Phanta DNA聚合酶进行扩增,得到线性化载体片段2;
将所述片段1和片段2连接起来,通过菌落PCR的方式进行验证得到pCRISPRi-low-GlxR质粒;
所述l-GlxR-F1的核苷酸序列如SEQ ID NO.8所示;
所述cas-R1的核苷酸序列如SEQ ID NO.3所示;
所述ori-F的核苷酸序列如SEQ ID NO.4所示;
所述CRISPRi-R2的核苷酸序列如SEQ ID NO.7所示;
2)5-氨基乙酰丙酸生产菌株构建及发酵:
将pCRISPRi-low-GlxR质粒通过电转导入谷氨酸棒杆菌CGS15-h3中,用菌落PCR的方式进行验证得到抑制全局转录调控因子基因glxR的表达菌株CGS15-l-GlxR;发酵,得到5-氨基乙酰丙酸。
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