CN113846132A - 苏氨酸生产菌种的构建及生产苏氨酸的方法 - Google Patents
苏氨酸生产菌种的构建及生产苏氨酸的方法 Download PDFInfo
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Abstract
本发明涉及微生物领域,特别涉及高转化率苏氨酸生产菌种的构建及生产苏氨酸的方法。本发明所述新型大肠杆菌L‑苏氨酸产量均高于各自的对照菌株,其中mqo表达活性增强的菌株摇瓶转化率在24‑30%之间,较出发菌提高6‑12个转化率;cyoABCDE表达活性增强的菌株摇瓶转化率在26‑30%之间,同样较出发菌株提高8‑12个转化率。有这两个位点改造的摇瓶结果可以得出,将苹果酸:醌氧化还原酶基因mqo的表达加强或者增强呼吸链末端氧化酶的活性均可以明显提高苏氨酸的生产能力。
Description
技术领域
本发明涉及微生物领域,特别涉及高转化率苏氨酸生产菌种的构建及生产苏氨酸的方法。
背景技术
L-苏氨酸是人和动物生长所必需的8种氨基酸之一,其广泛应用于饲料、食品添加及药物辅助材料制备等。目前L-苏氨酸主要通过微生物发酵生产,多种细菌可用于L-苏氨酸生产,如大肠杆菌、棒状杆菌属、沙雷氏菌属等的野生型诱导获得的突变株作为生产菌株。具体实例包括抗氨基酸类似物突变株或甲硫氨酸、赖氨酸、异亮氨酸等多种营养缺陷型。然而,传统诱变育种由于随机突变造成菌株生长缓慢及产生较多副产物,不易获得高产菌株。
随着全球苏氨酸需求量的不断增加,高产苏氨酸菌株的构建和改造尤为重要。现有技术中,有利用大肠杆菌,通过缺失苏氨酸操纵子序列的第-56至-18位的39bp序列,增强苏氨酸合成关键基因thrABC表达,苏氨酸生产力提高22%。KwangHoLee等利用系统代谢工程策略,通过突变编码天冬氨酸激酶I和III的基因thrA、lysC解除产物反馈抑制,通过敲除tdh及弱化ilvA来去除副产物甘氨酸、异亮氨酸,通过失活竞争途径基因metA和lysA为苏氨酸合成提供了更多前体等,最终获得的TH28C(pBRThrABCR3)菌株发酵50h可产酸82.4g/L,糖酸转化率39.3%。2016年梅花集团申请的中国专利201611248603.9中,通过强化pntAB、敲除iclR获得MHZ-0214-2菌株,该菌株苏氨酸产量为15.3g/L、转化率约为18.5%且无质粒负担。
发明内容
有鉴于此,本发明提供了进一步提高糖酸转化率的苏氨酸生产菌株的构建方法及生产苏氨酸的方法。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了强化mqo基因的表达和/或强化cyoABCDE基因的表达在生产L-苏氨酸中的应用。
在本发明的一些具体实施方案中,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。所述启动子强化包括选用强启动子;所述强启动子包括tac启动子。所述RBS序列强化为引入RBS序列。
本发明还提供了菌株,强化mqo基因的表达和/或强化cyoABCDE基因的表达。
在本发明的一些具体实施方案中,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。所述启动子强化包括选用强启动子;所述强启动子包括tac启动子。所述RBS序列强化为引入RBS序列。
在本发明的一些具体实施方案中,所述菌株的出发菌株为MHZ-0214-2,具体记载在公开号为106591209A的专利申请文件中。
本发明还提供了所述的菌株在生产L-苏氨酸中的应用。
本发明还提供了所述菌株的构建方法,强化mqo基因的表达和/或强化cyoABCDE基因的表达。
在本发明的一些具体实施方案中,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。所述启动子强化包括选用强启动子;所述强启动子包括tac启动子。所述RBS序列强化为引入RBS序列。本发明还提供了生产L-苏氨酸的方法,以所述的菌株为发酵菌株。
本发明所述新型大肠杆菌L-苏氨酸产量均高于各自的对照菌株,其中mqo表达活性增强的菌株摇瓶转化率在24-30%之间,较出发菌提高6-12个转化率;cyoABCDE表达活性增强的菌株摇瓶转化率在26-30%之间,同样较出发菌株提高8-12个转化率。有这两个位点改造的摇瓶结果可以得出,将苹果酸:醌氧化还原酶基因mqo的表达加强或者增强呼吸链末端氧化酶的活性均可以明显提高苏氨酸的生产能力。
两个位点同时改造的菌株,摇瓶转化率在35-52%之间,其苏氨酸的转化率率进一步提升。其中表现最好的菌株MHZ-0216-5的产量为44.6g/l,转化率为52.4%,出发菌MHZ-0214-2苏氨酸产量为15.3g/l,转化为18.0%,产量较出发菌提高191.50%,转化率提高191.35%,由此可见,改造菌的苏氨酸生产能力明显优于MHZ-0214-2。
具体实施方式
本发明公开了高转化率苏氨酸生产菌种的构建方法及生产苏氨酸的方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
根据大肠杆菌中L-苏氨酸的代谢路径,本发明以MHZ-0214-2为出发菌株(梅花集团专利申请公开号106591209A),在其基因组上进行相关改造,对苏氨酸代谢路径中关键基因进行相应的强化和过表达,如:将苹果酸:醌氧化还原酶基因mqo的表达加强,包括用强启动子替换原有启动子,或者增强RBS的强度以及在基因组上增加mqo的拷贝数;增加呼吸链末端氧化酶cyoABCDE的表达强度,包括用强启动子替换原有启动子,或者增强RBS的强度,以及在基因组上增加cyoABCDE的拷贝数;另外同时过表达两者,增加前体及能量的供应,来提高菌株生产苏氨酸的能力。
大肠杆菌的基因组编辑,主要借鉴了JiangY等报道的CRISPR-Cas9基因编辑技术(Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System,Jiang Y,Chen B,etal.Appl.Environ Microbiol,2015)。
以下实施例中,所述卡那霉素(Kanamycin)在培养基中的终浓度为50μg/mL,所述壮观霉素(spectinomycin)在培养基中的终浓度为50μg/ml。
以下实施例中,所用试剂均可由市场购得。本发明提供的高转化率苏氨酸生产菌种的亲代菌株为MHZ-0214-2,属于W3110(埃希氏菌属(Escherichia))。实施例中所使用引物序列见表1。
表1
本发明中涉及的基因名称解释如下:
thrA:天冬氨酸激酶及Ⅰ-高丝氨酸脱氢酶;
thrB:高丝氨酸激酶;
thrC:苏氨酸合成酶;
mqo:苹果酸:醌氧化还原酶;
tdcC:丝氨酸/苏氨酸:H+同向转运蛋白;
cyoABCDE:细胞色素bo3泛素氧化酶;
pyc:丙酮酸羧化酶;
本发明提供的高转化率苏氨酸生产菌种的构建及生产苏氨酸的方法中所用原料及试剂均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1:制备强化mqo基因的菌株MHZ-0214-3(启动子强化)
(1)pTarget-tac-mqo质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015),选用sgRNAmqotac-F/sgRNA-R引物对,扩增出带有N20的sgRNA片段①;Step2:以W3110基因组为模版,选用mqotac-UF/mqotac-UR引物对,扩增出含tac启动子的上游同源臂②;Step3:以W3110基因组为模版,选用mqotac-DF/mqotac-DR引物对,扩增出含tac启动子的下游同源臂③;Step4:以①、②、③为模板,选用sgRNAmqotac-F/mqotac-DR引物对,扩增出sgRNA-up-Ptac-down片段,也称Donor DNA;Step5:对DonorDNA进行SpeI和PstI双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶22℃进行连接。随后,转化Trans1-T1感受态细胞,获得pTarget-Ptac-mqo,并进行酶切鉴定。(2)感受态细胞制备及电转化pTarget-Ptac-mqo质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9System,JiangY,ChenB,et al.Appl.Environ Microbiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTarget-Ptac-mqo质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
用引物对mqotac-F/mqotac-R进行目的片段的扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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0.5mMIPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;Step2: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pTarget-Ptac-mqo质粒已丢失;Step3:挑取pTarget-Ptac-mqo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;Step4: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MHZ-0214-3(Ptac-mqo)菌株。
实施例2:制备RBS强化的mqo基因的菌株MHZ-0214-4(RBS-mqo)
(1)pTarget-RBS3-mqo质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015),选用sgRNAmqoRBS-F/sgRNA-R引物对,扩增出带有N20的sgRNA片段①;Step2:以W3110基因组为模版,选用mqoRBS-UF/mqoRBS-UR引物对,扩增出含RBS的上游同源臂②;Step3:以片段②为模版,选用mqoRBS-UF/mqoRBS-RBSR引物对,扩增出含RBS的上游同源臂③;Step4:以W3110基因组为模版,选用mqoRBS-DF/mqoRBS-DR引物对,扩增出含RBS的下游同源臂④;Step5:①、③、④为模版,选用sgRNAmqoRBS-F/mqoRBS-DR引物对,扩增出sgRNA-up-RBS-down片段,也称DonorDNA;Step6:对DonorDNA进行SpeI和BglII双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶22℃进行连接。随后,转化Trans1-T1感受态细胞,获得pTarget-RBS-mqo,并进行酶切鉴定。
(2)感受态细胞制备及电转化pTarget-RBS-mqo质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System,Jiang Y,Chen B,et al.Appl.EnvironMicrobiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTarget-RBS-mqo质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
Step1:使用引物对mqoRBS-RBSR/mqoRBS-UF对上述单菌落进行菌落PCR验证;Step2:将PCR鉴定正确的菌株用引物对mqoRBS-F/mqoRBS-R扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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;Step2: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pTarget-RBS-mqo质粒已丢失;Step3:挑取pTarget-RBS-mqo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;Step4: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MHZ-0214-4(RBS-mqo)菌株。
实施例3:制备强化mqo基因的菌株MHZ-0214-5(多拷贝)
(1)pTarget-zwf::mqo质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via theCRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015),选用sgRNAmqo2-F/sgRNA-R引物对,扩增出带有N20的sgRNA片段①;Step2:以W3110基因组为模版,选用mqo2-UF/mqo2-UR引物对,扩增出上游同源臂②;Step3:以W3110基因组为模版,选用mqo-F/mqo-R引物对,扩增出mqo片段③;Step4:以W3110基因组为模版,选用mqo2-DF/mqo2-DR引物对,扩增出含RBS的下游同源臂④;Step5:以①、②、③、④为模板,选用sgRNAmqo2-F/mqo2-DR引物对,扩增出sgRNA-up-mqo-down片段,也称DonorDNA;Step6:对DonorDNA进行SpeI和PstI双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶22℃进行连接。随后,转化Trans1-T1感受态细胞,获得pTarget-zwf::mqo,并进行酶切鉴定。
(2)感受态细胞制备及电转化pTarget-RBS3-mqo质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTarget-zwf::mqo质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
Step1:使用引物对mqo2-F/mqo2-R对上述单菌落进行扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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0.5mMIPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;Step2: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pTarget-zwf::mqo质粒已丢失;Step3:挑取pTarget-zwf::mqo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;Step4: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MHZ-0214-5(zwf::mqo)菌株。
实施例4:制备强化cyoABCDE基因的菌株MHZ-0214-6(启动子强化)
(1)pTarget-tac-cyoABCDE质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System,JiangY,ChenB,etal.Appl.Environ Microbiol,2015),选用sgRNAcyo-F/sgRNA-R引物对,扩增出带有N20的sgRNA片段①;Step2:以W3110基因组为模版,选用cyo-UF/cyo-UR引物对,扩增出上游同源臂②;Step3:以W3110基因组为模版,选用cyotac-F/cyo-DR引物对,扩增出含tac启动子的下游同源臂③;Step4:以③为模版,选用cyotac-DF/cyo-DR引物对,扩增出含tac启动子的下游同源臂④;Step5:以①、②、④为模板,选用sgRNAcyo-F/cyo-DR引物对,扩增出sgRNA-up-Ptac-down片段,也称DonorDNA;Step6:对Donor DNA进行SpeI和PstI双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶22℃进行连接。随后,转化Trans1-T1感受态细胞,获得pTarget-Ptac-cyoABCDE,并进行酶切鉴定。
(2)感受态细胞制备及电转化pTarget-Ptac-cyoABCDE质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System,JiangY,ChenB,et al.Appl.Environ Microbiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTarget-Ptac-cyoABCDE质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
用引物对cyo-F/cyo-R进行目的片段的扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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0.5mMIPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;Step2: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pTarget-Ptac-cyoABCDE质粒已丢失;Step3:挑取pTarget-Ptac-cyoABCDE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;Step4: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MHZ-0214-6(Ptac-cyoABCDE)菌株。
实施例5:制备cyoABCDE基因的RBS强化菌株
MHZ-0214-7(RBS::cyoABCDE)
(1)pTarget-RBS-cyoABCDE质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015),选用sgRNAcyo-F/sgRNA-R引物对,扩增出带有N20的sgRNA片段①;Step2:以W3110基因组为模版,选用cyo-UF/cyoRBS-UR引物对,扩增出含RBS的上游同源臂②;Step3:以W3110基因组为模版,选用cyoRBS-DF/cyo-DR引物对,扩增出含RBS的上游同源臂③;Step4:以①、②、③为模板,选用sgRNAcyo-F/cyo-DR引物对,扩增出sgRNA-up-RBS-down片段,也称DonorDNA;Step5:对Donor DNA进行SpeI和PstI双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶22℃进行连接。随后,转化Trans1-T1感受态细胞,获得pTarget-RBS-cyoABCDE,并进行酶切鉴定。
(2)感受态细胞制备及电转化pTarget-RBS-cyoABCDE质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.Environ Microbiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTarget-RBS-cyoABCDE质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
用引物对cyo-F/cyo-R扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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0.5mMIPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;Step2: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pTarget-RBS-cyoABCDE质粒已丢失;Step3:挑取pTarget-RBS-cyoABCDE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;Step4: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MHZ-0214-7(RBS::cyoABCDE)菌株。
实施例6:制备引入cyoABCDE基因的菌株MHZ-0214-8(多拷贝)
(1)pTarget-cyoABCDE质粒构建
Step1:以pTargetT质粒为模版(来源于文献Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System,JiangY,Chen B,etal.Appl.EnvironMicrobiol,2015),选用sgRNAcyo2-F/sgRNA-R引物对,扩增出带有N20的sgRNA的片段①;Step2:以W3110基因组为模版,选用cyo2-UF/cyo2-UR引物对,扩增出上游同源臂片段②;Step3:以W3110基因组为模版,选用cyo2-DF/cyo2-DR引物对,扩增出下游同源臂③;Step4:以W3110基因组为模版,选用cyo-F/cyo-R引物对,扩增出cyoABCDE片段④;Step5:对①、②、③、④进行融合PCR扩增得到完整片段sgRNA-Up arm-cyoABCDE-Down arm;Step6:对Donor DNA进行SpeI和PstI双酶切,并将其与载体片段(双酶切并去磷酸化)按照合适的比例用T4连接酶23℃进行连接,转化Trans1-T1感受态细胞,获得pTargetT-cyoABCDE。
(2)感受态细胞制备及电转化pTarget-cyoABCDE质粒
Step1:将pCas质粒(来源于文献Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System,Jiang Y,Chen B,et al.Appl.EnvironMicrobiol,2015)电转入MHZ-0214-2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》);Step2:挑取MHZ-0214-2(pCas)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OD650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》)。Step3:将pTargetT-cyoABCDE质粒电转入MHZ-0214-2(pCas)感受态细胞中(电转化条件: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
Step1:将PCR鉴定正确的菌株用引物对cyo2-F/cyo2-R扩增,扩增产物送测序,以验证序列的完整性。
(4)构建相关质粒丢失
Step1: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0.5mMIPTG的LB试管中,30℃过夜培养后划线于含卡那霉素的LB平板上;Step2: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-cyoABCDE质粒已丢失;Step3:挑取pTargetT-cyoABCDE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;Step4: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
MHZ-0214-8(tdcC::cyoABCDE)菌株。
实施例7:制备强化mqo基因并同时引入cyoABCDE基因的菌株
按照实施例1-6的方法,对mqo和cyoABCDE两个位点进行不同改造方式的叠加,共获得9株改造菌。
实施例1-7所获得的产苏氨酸基因改造菌株如表2所示。
表2本发明构建的基因工程菌
实施例8:产L-苏氨酸基因工程菌摇瓶发酵验证
Step1:从冻存管中取MHZ-0214-2、MHZ-0214-3、MHZ-0214-4、MHZ-0214-5、MHZ-0214-6、MHZ-0214-7、MHZ-0214-8、MHZ-0216-0、MHZ-0216-1、MHZ-0216-2、MHZ-0216-3、MHZ-0216-4、MHZ-0216-5、MHZ-0216-6、MHZ-0216-7、MHZ-0216-8共16株,在LB平板上划线活化,37℃培养18-24h;Step2:将菌体从平板上刮下一环,接种到装有50mL种子培养基(见表3)的摇瓶中,37℃,转速90rpm,培养约5小时,使OD650控制在2以内;Step3:将2mL种子液转接到含20mL发酵培养基(见表4)的摇瓶中,往复摇床37℃,100rpm发酵培养直至残糖耗尽,发酵结束后测定样品OD650,并用使用HPLC测定L-苏氨酸含量,用生物传感仪方法测定残糖量。为了确保实验的可靠性,对摇瓶进行3次重复实验,其产酸及转化率结果的平均值见表5。
表3种子培养基(g/L)
成分 | 浓度 |
葡萄糖 | 25 |
玉米浆 | 25 |
豆粕水解液 | 7.7 |
酵母膏 | 2.5 |
KH<sub>2</sub>PO<sub>4</sub> | 1.4 |
七水硫酸镁 | 0.5 |
FeSO<sub>4</sub>、MnSO<sub>4</sub> | 20mg/L |
pH | 7.0 |
表4发酵培养基(g/L)
成分 | 浓度 |
葡萄糖 | 85 |
玉米浆 | 6 |
豆粕水解液 | 7.7 |
七水硫酸镁 | 0.5 |
KH<sub>2</sub>PO<sub>4</sub> | 1.0 |
天冬氨酸 | 10 |
FeSO<sub>4</sub>、MnSO<sub>4</sub> | 30mg/L |
生物素 | 50μg |
硫胺素 | 500μg |
pH | 7.2 |
表5产苏氨酸基因工程菌生产力比较
注:*表示P值<0.01,说明其与对照相比有明显差异
由表5可知,本发明所述新型大肠杆菌L-苏氨酸产量均高于各自的对照菌株,其中mqo表达活性增强的菌株摇瓶转化率在24-30%之间,较出发菌提高6-12个转化率;cyoABCDE表达活性增强的菌株摇瓶转化率在26-30%之间,同样较出发菌株提高8-12个转化率。有这两个位点改造的摇瓶结果可以得出,将苹果酸:醌氧化还原酶基因mqo的表达加强或者增强呼吸链末端氧化酶的活性均可以明显提高苏氨酸的生产能力。
两个位点同时改造的菌株,摇瓶转化率在35-52%之间,其苏氨酸的转化率率进一步提升。其中表现最好的菌株MHZ-0216-5的产量为44.6g/l,转化率为52.4%,出发菌MHZ-0214-2苏氨酸产量为15.3g/l,转化为18.0%,产量较出发菌提高191.50%,转化率提高191.35%,由此可见,改造菌的苏氨酸生产能力明显优于MHZ-0214-2。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 苏氨酸生产菌种的构建及生产苏氨酸的方法
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Claims (8)
1.强化mqo基因的表达和/或强化cyoABCDE基因的表达在生产L-苏氨酸中的应用。
2.如权利要求1所述的应用,其特征在于,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。
3.菌株,其特征在于,强化mqo基因的表达和/或强化cyoABCDE基因的表达。
4.如权利要求3所述的菌株,其特征在于,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。
5.如权利要求3或4所述的菌株在生产L-苏氨酸中的应用。
6.如权利要求3或4所述菌株的构建方法,其特征在于,强化mqo基因的表达和/或强化cyoABCDE基因的表达。
7.如权利要求6所述的构建方法,其特征在于,所述强化包括启动子强化、RBS序列强化或增加拷贝数中的一种或多种的组合。
8.生产L-苏氨酸的方法,其特征在于,以如权利要求3或4所述的菌株为发酵菌株。
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CN114875090B (zh) * | 2022-05-31 | 2024-03-26 | 廊坊梅花生物技术开发有限公司 | 一种生产赖氨酸的方法及应用 |
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