CN116536292A - 磷酸葡萄糖酸脱水酶突变体及其应用 - Google Patents
磷酸葡萄糖酸脱水酶突变体及其应用 Download PDFInfo
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Abstract
本发明提出了磷酸葡萄糖酸脱水酶突变体及其应用,属于基因工程技术领域。所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为苯丙氨酸和434位氨基酸由苏氨酸替换为异亮氨酸,其氨基酸序列如SEQ ID NO.1,所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为赖氨酸和434位氨基酸由苏氨酸替换为谷氨酸,其氨基酸序列如SEQ ID NO.3。本发明提供一种磷酸葡萄糖酸脱水酶变体,该变体可精微地扰动代谢平衡,并有利于提高肠杆菌的赖氨酸的合成效率。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及磷酸葡萄糖酸脱水酶突变体及其应用。
背景技术
L-赖氨酸是碱性必需氨基酸,分子式为C6H14N2O2,外观为白色或近乎于白色结晶粉末。在210℃变暗,在224.5℃下分解,易溶于水,微溶于醇,不溶于醚。广泛应用于动物饲料、医药和食品工业,其中L-赖氨酸约90%用于饲料工业,10%用于食品和医药行业。用于动物饲料添加剂,L-赖氨酸可帮助机体吸收其他氨基酸,从而提高饲料质量。因此,生产赖氨酸具有广阔的前景。
目前,L-赖氨酸最常用的生产方法为微生物发酵法,微生物发酵法具有原料成本低、反应条件温和、容易实现大规模生产等优点。大肠杆菌(Escherichia coli)由于其生长速度快,遗传背景清晰,培养条件简单,代谢工程手段成熟等优点,广泛应用于工业发酵领域,可用于生产L-氨基酸、核苷酸及其他有机酸等。
大肠杆菌合成L-赖氨酸,从L-天冬氨酸开始,经过多步酶催化反应得到L-赖氨酸。L-赖氨酸的产率与生物合成途径中的酶活性及代谢流的分布相关,通过增强末端合成途径的酶活性,或改变中央代谢流方向,以及增加还原力的供给,都利于大肠杆菌高产L-赖氨酸。在大肠杆菌中,ED途径作为降解葡萄糖的主要途径之一,其中葡萄糖经HMP途径的前部分生成6-磷酸葡糖酸后,不再氧化脱羧,而是在磷酸葡萄糖酸脱水酶(phosphogluconatedehydratase,EDD)的催化下进行脱水,生成1-酮-3-脱氧-6-磷酸葡糖酸,再由脱氧酮糖酸醛缩酶催化裂解为3-磷酸甘油醛和丙酮酸,3-磷酸甘油醛可进入EMP途径转变成丙酮酸,丙酮酸可被催化为赖氨酸的前体草酰乙酸。
但至今尚未有相关报道,ED途径的基因或酶的变化对赖氨酸的合成有影响。本发明的发明人在多年的研究和实践中发现,ED途径的磷酸葡萄糖酸脱水酶可微妙的调控赖氨酸的合成。该酶的一些特定的变体,可以显著地提高肠杆菌对赖氨酸的合成效率,从而完成了本发明。
发明内容
本发明的目的在于提出磷酸葡萄糖酸脱水酶突变体及其应用,具有良好的抗衰老的效果。
本发明的技术方案是这样实现的:
本发明提供一种磷酸葡萄糖酸脱水酶突变体,所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为苯丙氨酸和434位氨基酸由苏氨酸替换为异亮氨酸,其氨基酸序列如SEQ ID NO.1。
本发明提供一种编码上述磷酸葡萄糖酸脱水酶突变体的基因,其核苷酸序列如SEQ ID NO.2。
本发明提供一种磷酸葡萄糖酸脱水酶突变体,所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为赖氨酸和434位氨基酸由苏氨酸替换为谷氨酸,其氨基酸序列如SEQ ID NO.3。
本发明提供一种编码上述磷酸葡萄糖酸脱水酶突变体的基因,其核苷酸序列如SEQ ID NO.4。
本发明提供一种包含上述磷酸葡萄糖酸脱水酶突变体的基因的工程菌。
作为本发明的进一步改进,所述工程菌为大肠杆菌。
作为本发明的进一步改进,所述工程菌为赖氨酸生产菌株,保藏编号为CGMCCNo.22648。
本发明进一步保护一种上述工程菌在生产赖氨酸中的应用。
本发明具有如下有益效果:本发明提供一种磷酸葡萄糖酸脱水酶变体,该变体可精微地扰动代谢平衡,并有利于提高肠杆菌的赖氨酸的合成效率。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
赖氨酸生产菌株的保藏编号为CGMCC No.22648,于2021年6月1日保藏在中国微生物菌种保藏管理委员会普通微生物中心,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,分类命名为:大肠埃希菌Escherichia coli。
术语解释:
本发明中涉及的基因名称解释如下:
Phosphogluconatedehydratase:磷酸葡萄糖酸脱水酶;edd:磷酸葡萄糖酸脱水酶编码基因。
模式菌MG1655为MG1655 E.coli Strain,购于上海泽叶生物科技有限公司。
以下实施例中使用的引物序列信息如表1所示:
表1引物序列信息
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购买得到的常规产品。
实施例1在模式菌MG1655中引入磷酸葡萄糖酸脱水酶变体eddS379F、T434I
(1)pTargetT-N20-edd质粒及Donor DNA构建
步骤1:使用pTF-edd-sgRNA-F/pTF-edd-sgRNA-R为引物,以质粒pTargetT为模板(可参见Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9System,Jiang Y,ChenB,etal.Appl.EnvironMicrobiol,2015),扩增出带有N20的pTF线性质粒,使用无缝组装Clone Express试剂盒将此线性质粒37℃进行组装,随后转化Trans1-T1感受态细胞,获得pTargetT-N20-edd,并进行PCR鉴定及测序验证;
步骤2:以MG1655基因组为模板,选用edd-S379F、T434I-UF/edd-S379F、T434I-UR引物对,扩增出上游同源臂①,选用edd-S379F、T434I-DF/edd-S379F、T434I-DR引物对,扩增出下游同源臂②,选用edd-S379F、T434I-MF/edd-S379F、T434I-MR引物对,扩增出中间同源臂③,以①、②、③为模板,选用edd-S379F、T434I-UF/edd-S379F、T434I-DR引物对,扩增出Donor DNA,Donor DNA的作用是提供同源重组片段。
(2)感受态细胞制备及电转化
步骤1:将pCas质粒(可参见Multigene Editing in the EscherichiacoliGenome via the CRISPR-Cas9 System,JiangY,ChenB,etal.Appl.EnvironMicrobiol,2015)电转入模式株MG1655的感受态细胞中(转化方法及感受态制备方法均参照《分子克隆III》)。
步骤2:挑取单菌落于5mL含卡那霉素和终浓度为10mM阿拉伯糖的LB试管中,30℃,200r/min培养至OD 650为0.4后制备电转感受态细胞(感受态制备方法参照《分子克隆III》)。
步骤3:将(1)中构建得到的pTargetT-N20-edd质粒和Donor DNA同时电转入带有pCas的感受态细胞中(电转条件:2.5kV,200Ω,25μF),涂布于含壮观霉素和卡那霉素的LB平板上,30℃静置培养至单菌落可见。
(3)重组验证
步骤1:使用引物对edd-S379F、T434I-F1/edd-S379F、T434I-DR和edd-S379F、T434I-F2/edd-S379F、T434I-DR对上述单菌落进行菌落PCR验证是否同时存在两个突变点;步骤2:将PCR鉴定正确的菌株用引物对edd-S379F、T434I-UF/edd-S379F、T434I-DR扩增,扩增产物送测序,以验证序列的完整性。
(4)质粒的消除
步骤1:挑取测序验证正确的单菌落接种于5mL含卡那霉素及终浓度为0.5mM IPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;
步骤2:挑取单菌落对点于含卡那霉素、壮观霉素LB平板和只含卡那霉素的LB平板上,30℃过夜培养,若在含卡那霉素、壮观霉素的LB平板上不能生长,在卡那霉素的LB平板上生长,表明pTargetT-N20质粒已成功消除;
步骤3:挑取pTargetT-N20质粒成功消除的阳性菌落,接种于无抗LB试管中,42℃培养8h后划线于LB平板上,37℃过夜培养;
步骤4:挑取单菌落对点于含卡那霉素LB平板和无抗LB平板上,若在含卡那霉素的LB平板上不能生长,在无抗LB平板上生长,表明pCas质粒丢失,得到的菌株命名为MG1655-eddS379F、T434I。
实施例2在菌株CGMCC No.22648中引入磷酸葡萄糖酸脱水酶变体eddS379F、T434I
实施例2所用到的方法同实施例1,包括工具质粒、Doner DNA、感受态制备和基因重组的筛选方法,区别在于目标改造菌的替换。最后获得的菌株命名为CGMCC No.22648-eddS379F、T434I。
实施例3在模式菌MG1655中引入磷酸葡萄糖酸脱水酶变体eddS379K、T434E
构建原理及方法同实施例1,获得含有目的突变的菌株,其磷酸葡萄糖酸脱水酶变体第379位氨基酸由丝氨酸替换为赖氨酸(S379K)和434位氨基酸由苏氨酸替换为谷氨酸(T434E),获得的菌株命名为MG1655-eddS379K、T434E。
(1)Donor DNA构建
步骤1:以MG1655基因组为模板,选用edd-S379F、T434I-UF/edd-S379F、T434I-UR引物对,扩增出上游同源臂①,选用edd-S379F、T434I-DF/edd-S379F、T434I-DR引物对,扩增出下游同源臂②,选用edd-S379K、T434E-MF/edd-S379K、T434E-MR引物对,扩增出中间同源臂③,以①、②、③为模板,选用edd-S379F、T434I-UF/edd-S379F、T434I-DR引物对,扩增出Donor DNA。
(2)感受态细胞制备及电转化
步骤1:制备MG1655的感受态细胞,将pCas质粒电转入该感受态细胞中(转化方法及感受态制备方法均参照《分子克隆III》)。
步骤2:挑取单菌落于5mL含卡那霉素和终浓度为10mM阿拉伯糖的LB试管中,30℃,200r/min培养至OD 650为0.4后制备电转感受态细胞(感受态制备方法参照《分子克隆III》)。
步骤3:将实施例1中构建得到的pTargetT-N20-edd质粒和Donor DNA同时电转入带有pCas的感受态细胞中(电转条件:2.5kV,200Ω,25μF),涂布于含壮观霉素和卡那霉素的LB平板上,30℃静置培养至单菌落可见。
(3)重组验证
步骤1:使用引物对edd-S379K、T434E-F1/edd-S379F、T434I-DR和edd-S379K、T434E-F2/edd-S379F、T434I-DR对上述单菌落进行菌落PCR验证是否同时存在两个突变点;步骤2:将PCR鉴定正确的菌株用引物对edd-S379F、T434I-UF/edd-S379F、T434I-DR扩增,扩增产物送测序,以验证序列的完整性。
(4)质粒的消除
步骤1:挑取测序验证正确的单菌落接种于5mL含卡那霉素及终浓度为0.5mM IPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;
步骤2:挑取单菌落对点于含卡那霉素、壮观霉素LB平板和只含卡那霉素的LB平板上,30℃过夜培养,若在含卡那霉素、壮观霉素的LB平板上不能生长,在卡那霉素的LB平板上生长,表明pTargetT-N20质粒已丢失;
步骤3:挑取pTargetT-N20质粒丢失的阳性菌落,接种于无抗LB试管中,42℃培养8h后划线于LB平板上,37℃过夜培养;
步骤4:挑取单菌落对点于含卡那霉素LB平板和无抗LB平板上,若在含卡那霉素的LB平板上不能生长,在无抗LB平板上生长,表明pCas质粒丢失,得到的菌株命名为MG1655-eddS379K、T434E。
实施例4、在菌株CGMCC No.22648中引入磷酸葡萄糖酸脱水酶变体eddS379K、T434E
实施例4所用到的方法同实施例3,包括工具质粒、Doner DNA、感受态制备和基因重组的筛选方法,区别在于目标改造菌的替换。最后获得的菌株命名为CGMCC No.22648-eddS379K、T434E。
实施例5重组菌株生产赖氨酸的性能
培养基:
活化培养基:蛋白胨10g/L,NaCl 10g/L,酵母粉5g/L,18g/L琼脂粉,调节pH至7.0。
种子培养基:葡萄糖10g/L,硫酸铵4g/L,酵母膏2.0g/L,磷酸二氢钾3g/L,七水硫酸镁0.4g/L,硫酸亚铁0.01g/L,硫酸锰0.01g/L,pH7.0。
发酵培养基:葡萄糖50g/L,硫酸铵25g/L,酵母膏4.0g/L,磷酸二氢钾1.6g/L,七水硫酸镁1.0g/L,硫酸亚铁0.03g/L,硫酸锰0.03g/L,碳酸钙25,pH7.0。
性能验证方法:
(1)种子活化:从冻存管中取待验证菌株,在种子活化培养基上划线活化,37℃培养12h;
(2)种子培养:挑取平板活化种子1环接至装有20mL种子培养基的500mL三角瓶中,33℃,220r/min振荡培养7h;
(3)发酵培养:将2mL种子液接种至装有20mL发酵培养基的500mL三角瓶中,33℃、220r/min振荡培养12h。
(4)取2mL发酵液离心(12000rpm,2min),收集上清液,用HPLC检测重组菌与对照菌发酵液中的L-赖氨酸含量,其检测结果如下表2表示(表中数据为三批次平均值)。
表2重组菌株生产赖氨酸的性能测试
发酵结果可知,本发明的2种重组菌株相比模式菌MG1655和CGMCC No.22648,其赖氨酸产量及转化率均有不同程度的提高,说明本发明的突变位点有正效果。
磷酸葡萄糖酸脱水酶的2种突变体均有正效果,但具有S379K、T434E突变的效果更好,其CGMCC No.22648-eddS379K、T434E赖氨酸转化率相比高产赖氨酸菌CGMCC No.22648提高了6.2%
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种磷酸葡萄糖酸脱水酶突变体,其特征在于,所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为苯丙氨酸和434位氨基酸由苏氨酸替换为异亮氨酸,其氨基酸序列如SEQ ID NO.1。
2.编码权利要求1所述磷酸葡萄糖酸脱水酶突变体的基因,其特征在于,其核苷酸序列如SEQ ID NO.2。
3.一种磷酸葡萄糖酸脱水酶突变体,其特征在于,所述磷酸葡萄糖酸脱水酶突变体其379位氨基酸由丝氨酸替换为赖氨酸和434位氨基酸由苏氨酸替换为谷氨酸,其氨基酸序列如SEQ ID NO.3。
4.编码权利要求1所述磷酸葡萄糖酸脱水酶突变体的基因,其特征在于,其核苷酸序列如SEQ ID NO.4。
5.一种包含权利要求2或4所述磷酸葡萄糖酸脱水酶突变体的基因的工程菌。
6.根据权利要求5所述的工程菌,其特征在于,所述工程菌为大肠杆菌。
7.根据权利要求5所述的工程菌,其特征在于,所述工程菌为赖氨酸生产菌株,保藏编号为CGMCC No.22648。
8.一种如权利要求5-7任一项所述工程菌在生产赖氨酸中的应用。
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