CN107142234A - 一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法 - Google Patents
一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法 Download PDFInfo
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- CN107142234A CN107142234A CN201710336026.7A CN201710336026A CN107142234A CN 107142234 A CN107142234 A CN 107142234A CN 201710336026 A CN201710336026 A CN 201710336026A CN 107142234 A CN107142234 A CN 107142234A
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- corynebacterium glutamicum
- lysc
- glutamicum
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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Abstract
本发明提供了一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法。本发明重组谷氨酸棒杆菌通过在谷氨酸棒杆菌中过表达解除反馈抑制的天冬氨酸激酶基因lysC,再置换该重组菌中的二氢嘧啶二羧酸合成酶的启动子以弱化二氢嘧啶二羧酸合成酶的活性,然后将四氢嘧啶合成途径相关基因ectABC转入重组菌得到。本发明的重组谷氨酸棒杆菌能够利用不同的廉价原料在低盐条件下发酵生产四氢嘧啶,还可以使用廉价的玉米浆作为营养成分替代昂贵的酵母粉,可以进一步降低原料的成本,同时本发明的重组谷氨酸棒杆菌解决了生物安全性问题,简化了后提取工艺,具有良好的市场应用前景。
Description
技术领域
本发明属于基因工程和生物发酵技术领域,具体地说,涉及一种利用食品安全级的微生物重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法。
背景技术
四氢嘧啶(Ectoine)是一种环状的氨基酸衍生物,是许多耐盐微生物为维持渗透压平衡而在细胞内产生的一种相容性溶质,对于细胞/酶的抗逆性具有重要的作用,可以缓解高渗、高温、冻融、干燥、辐射和化学试剂对蛋白、核酸、生物膜及整个细胞的毒害作用。因此四氢嘧啶目前已被广泛地应用于化妆品、医药、酶制剂领域,具有广阔的市场前景。
目前,四氢嘧啶的生产方法主要通过嗜盐微生物特别是盐单胞菌的高密度发酵来获得。这个过程通过采用被称为“细菌挤奶法”的方式进行生产,即先在高渗透压下培养细胞并在胞内积累四氢嘧啶,然后通过低渗冲击刺激四氢嘧啶从胞内释放到胞外,然后再重复进行高渗培养、低渗冲击,以获得较高浓度的四氢嘧啶。这个过程操作繁杂,且需要在高盐条件下进行培养,因此对设备要求高,导致其生产价格十分昂贵。
目前正在开发的一些工艺采用重组大肠杆菌进行发酵,以葡萄糖或者天冬氨酸为底物在低盐的条件下即可获得较高浓度的四氢嘧啶,简化了四氢嘧啶的生产工艺。然而,大肠杆菌会分泌内毒素,而四氢嘧啶主要应用于化妆品及医药领域,因此必须去除内毒素,使得整个分离过程较为复杂,增加了生产成本。因此开发安全高效的重组微生物使其能够在低盐的条件下连续生产四氢嘧啶且不会分泌内毒素,具有重要的应用价值。
发明内容
本发明的目的是提供一种生物安全度高、产量高、操作简便且成本低廉的利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法,以克服现有技术的不足。
四氢嘧啶的生物合成途径如图1所示。本发明主要改造方案为:在谷氨酸棒杆菌中过表达解除反馈抑制的天冬氨酸激酶基因lysC(序列如Seq ID1所示),强化流向天冬氨酸途径的碳流量,该基因包含定点突变Q298G且包含一个强启动子P1;通过置换二氢嘧啶二羧酸合成酶的启动子(序列如SeqID2所示)弱化二氢嘧啶二羧酸合成酶的活性,降低流向赖氨酸的碳流量;过表达四氢嘧啶合成途径相关基因ectABC(序列如Seq ID1所示);将该菌株在发酵罐中进行发酵培养获得四氢嘧啶。
本发明首先提供一种重组谷氨酸棒杆菌,含有突变后的lysC基因及启动子,突变后的dapA基因及启动子和ectABC基因;
所述突变后的lysC基因及启动子的序列如SEQ ID NO.1所示;
所述突变后的dapA基因及启动子的序列如SEQ ID NO.2所示;
所述ectABC基因的序列如SEQ ID NO.3所示。
本发明的重组谷氨酸棒杆菌,通过以下方法制备得到:
(1)将解除反馈抑制的天冬氨酸激酶基因lysC和强启动子片段P1连接到自杀性质粒上,获得的重组质粒,将该重组质粒电转化入谷氨酸棒杆菌中,得到重组菌C.glutamicumlysC-P1-298;
(2)通过启动子置换弱化重组菌C.glutamicum lysC-P1-298中的二氢嘧啶二羧酸合成酶基因dapA的表达,得到重组菌C.glutamicum lysC-dap;
(3)在重组菌C.glutamicum lysC-dap中转入能够过表达四氢嘧啶合成途径相关基因ectABC的表达载体,构建得到重组菌C.glutamicum lysC-dap-ectABC。
其中,步骤(1)是将lysC1,lysC2,lysC3和强启动子片段P1连接到自杀性质粒上,所述lysC1,lysC2,lysC3分别通过引物对SEQ ID NO.5-6,SEQ ID NO.7-8,SEQ ID NO.9-10以谷氨酸棒杆菌基因组为模板PCR扩增得到。
所述强启动子片段P1,其序列如SEQ ID NO.4所示,所述自杀性质粒为pK18mobsacB。
步骤(2)是将dap1和dap2片段连接到自杀性质粒上,获得的重组质粒;
所述dap1和dap2片段分别通过引物对SEQ ID NO.11-12,SEQ ID NO.13-14以谷氨酸棒杆菌基因组为模板PCR扩增得到。
步骤(3)是将ectABC基因连接到表达载体pXMJ19上,获得的重组质粒命名为pXMJ-ectABC,将该重组质粒电转化入步骤(2)的重组菌C.glutamicum lysC-dap中;所述ectABC基因通过引物对SEQ ID NO.15-16以Halomonas elongata DSM 2581(购自DSMZ)基因组为模板PCR扩增得到。
本发明的重组谷氨酸棒杆菌的培养基中含有氯霉素。具体地,含有5mg/L氯霉素。
本发明提供了重组菌C.glutamicum lysC-dap-ectABC在生产四氢嘧啶中的应用。
本发明提供了利用权利要求重组谷氨酸棒杆菌C.glutamicum lysC-dap-ectABC发酵生产四氢嘧啶的方法,以含可发酵糖的原料为底物,接入重组谷氨酸棒杆菌C.glutamicum lysC-dap-ectABC,发酵温度28-40℃,pH值5-8,溶氧值10%进行发酵。
所述含可发酵糖的原料为糖蜜,蔗糖、葡萄糖、淀粉水解液、玉米浆、木糖、甘露糖或甘油;发酵温度为32-35℃,pH值6-7,在发酵3-5h时添加终浓度为0.01-1mM的IPTG。
发酵过程中流加碳源,以及发酵培养基中含有硫胺素和生物素。
谷氨酸棒杆菌是一种食品安全级的微生物,广泛应用于氨基酸的生产,比如谷氨酸和赖氨酸的生产。谷氨酸棒杆菌不会分泌内毒素,因此利用谷氨酸棒杆菌来发酵生产四氢嘧啶解决了生物安全性的问题,大大简化了后提取工艺。本发明所构建的重组谷氨酸棒杆菌能够利用葡萄糖等廉价原料在低盐的条件下连续发酵生产四氢嘧啶,大大简化了四氢嘧啶的发酵过程。同时谷氨酸棒杆菌能够利用不同的廉价原料进行发酵,例如利用废弃糖蜜、蔗糖、淀粉水解液等,同时还可以使用廉价的玉米浆作为营养成分替代昂贵的酵母粉,因此可以进一步降低原料的成本。同时本发明的重组谷氨酸棒杆菌解决了生物安全性问题,简化了后提取工艺,具有良好的市场应用前景。
附图说明
图1为四氢嘧啶的生物合成途径以及本发明的主要改造位点。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1过表达解除反馈抑制的天冬氨酸激酶基因lysC
四氢嘧啶的直接合成前体是天冬氨酸,从天冬氨酸到四氢嘧啶合成途径的第一个酶天冬氨酸激酶受到赖氨酸和苏氨酸的反馈抑制。因此,本发明首先通过在天冬氨酸激酶基因中引入定点突变Q298G来解除以赖氨酸和苏氨酸对该酶的反馈抑制,并在该基因(lysC)前插入一个强启动子P1(序列为5’-ggtgcacaaagCAAAAGCTGGGTACCTCTATCTGGTGCCCTAAACGGGGGAATATTAACGGGCCCAGGGTGGTCGCACCTTGGTTGGTAGGAGTAGCATGGGATCC-)(SEQ ID NO.4)来强化该基因的表达。具体的实施过程如下:
以谷氨酸棒杆菌MB001(Baumgart M et al,Appl Environ Microbiol 79:6006-15(2013))的基因组为模板,以引物lysC-1-F(aggaaacagctatgacatgattacgtttcgcccagaaccaagtagcc)和lysC-1-R(CCCAGCTTTTGctttgtgcacctttcgatctacgt)为引物进行PCR,获得基因片段lysC1约1.0kb并进行PCR产物纯化。以谷氨酸棒杆菌MB001的基因组为模板,以引物lysC-2-F(CATGGGATCCatggccctggtcgtacag aa)和lysC-2-R(gagacgttgcccagaaccatgtcaatgttgatttctgca)为引物进行PCR,获得基因片段lysC2约0.9kb并进行PCR产物纯化。以谷氨酸棒杆菌MB001的基因组为模板,以引物lysC-3-F(acatggttctgggcaacgtctcttctgtagaagacg)和lysC-3-R(tgcatgcctgcaggtcgactacataaggtccgacatcgcct)为引物进行PCR,获得基因片段lysC3约1.0kb并进行PCR产物纯化。以本实验室构建的质粒pEC-yqhD-pduCDEGH(Huang et al.,Scientific Reports 7:42246,2017)为模板,以引物P1-F(ggtgcacaaagCAAAAGCTGGGTACCTCTATCTGGT)和P1-R(ccagggccatGGATCCCATGCTACTCCT)为引物进行PCR,获得基因片段P1约0.1kb并进行PCR产物纯化。将谷氨酸棒杆菌自杀性质粒pK18mobsacB(Journal of Biotechnology 104(2003)287-299)用EcoRI/XbaI进行双酶切,利用Gibson Assembly试剂盒(NEB)将lysC1,lysC2,lysC3和P1片段一步连接到pK18mobsacB上,获得的重组质粒命名为pK18-lysC。利用电穿孔仪(伯乐)将pK18-lysC通过电转化转入到谷氨酸棒杆菌MB001中,电击条件为电压2.5KV,电阻200Ω,电容25μF(电击杯宽度为2mm),通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养,之后在含有100g/L的蔗糖LB平板上进行二次筛选。利用P1-1-F和lysC-3-R为引物进行菌落PCR,正确的重组子能克隆出约2Kb的片段,该重组菌命名为C.glutamicum lysC-P1-298。该重组菌的关键特征是包括突变后的lysC基因及启动子,序列SEQ ID NO.1所示,在该核酸序列的第998-1000位碱基发生定点突变Q298G。该重组菌可以利用80g/L的葡萄糖产18g/L的赖氨酸,而野生菌株MB001不能产赖氨酸,说明在本实施例中,lysC基因修饰是成功的,即葡萄糖能够流向赖氨酸和四氢嘧啶共同的代谢支路。具体的发酵过程见实施例4。
实施例2启动子置换弱化二氢嘧啶二羧酸合成酶基因dapA的表达
由于重组菌C.glutamicum lysC-P1-298的主要发酵产物是赖氨酸,为了使更多的底物用于四氢嘧啶的合成,进一步弱化二氢嘧啶二羧酸合成酶基因dapA的表达。
以谷氨酸棒杆菌MB001的基因组为模板,以引物dap-1-F(ctatgacatgattacgaattcagatggttttcctgaccagctt)和dap-1-R(gggaagaaggaaaccttgaactctatgagcacagg)为引物进行PCR,获得基因片段dap1约1.0kb并进行PCR产物纯化。以谷氨酸棒杆菌MB001的基因组为模板,以引物dap-2-F(ttcaaggtttccttcttccctcatttggggg)和dap-2-R(tgcctgcaggtcgactctagaggcctgtaaaggctcatttcag)为引物进行PCR,获得基因片段dap2约1.0kb并进行PCR产物纯化。将谷氨酸棒杆菌自杀性质粒pK18mobsacB(Journal ofBiotechnology 104(2003)287-299)用EcoRI/XbaI进行双酶切,利用Gibson Assembly试剂盒(NEB)将dap1和dap2片段一步连接到pK18mobsacB上,获得的重组质粒命名为pK18-dap。利用电穿孔仪(伯乐)将pK18-dap通过电转化转入到谷氨酸棒杆菌C.glutamicum lysC-P1-298中,电击条件为电压2.5KV,电阻200Ω,电容25μF(电击杯宽度为2mm),通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养,之后在含有100g/L的蔗糖LB平板上进行二次筛选。利用dap-1-F和dap-2-R为引物进行菌落PCR并进行测序,正确的重组菌命名为C.glutamicum lysC-dap。该重组菌的关键特征是包括突变后的dapA基因及启动子,序列SEQ ID NO.2所示。该重组菌可以利用80g/L的葡萄糖产6g/L的赖氨酸同时生产2g/L的甘氨酸。具体的发酵过程见实施例4。
实施例3过表达四氢嘧啶合成途径相关基因ectABC
为了强化表达四氢嘧啶合成途径的关键基因,人工合成了ectABC基因,序列如SEQID NO.3所示。将Halomonas elongata DSM2581基因组以引物ectABC-F(tgcatgcctgcaggtcgactAGGAGGCCCTTCAGatgaacg)和ectABC-R(ccgccaaaacagccaagctgttacagcggcttctggtcgt)为引物进行PCR,获得基因片段ectABC约2.5kb并进行PCR产物纯化。将谷氨酸棒杆菌表达质粒pXMJ19(购置addgene)用EcoRI/XbaI进行双酶切,利用Gibson Assembly试剂盒(NEB)将ectABC基因片段一步连接到pXMJ19上,获得的重组质粒命名为pXMJ-ectABC。利用电穿孔仪(伯乐)将pXMJ-ectABC通过电转化转入到谷氨酸棒杆菌C.glutamicum lysC-dap中,电击条件为电压2.5KV,电阻200Ω,电容25μF(电击杯宽度为2mm),在含5mg/L的氯霉素LB平板上筛选重组菌,该重组菌命名为C.glutamicum lysC-dap-ectABC。该重组菌的关键特征是包括ectABC基因,序列SEQ IDNO.3所示。该重组菌可以利用80g/L的葡萄糖产12g/L的四氢嘧啶,而实施例1和2中的菌均不能产四氢嘧啶。具体的发酵过程见实施例4。
实施例4重组谷氨酸棒杆菌C.glutamicum lysC-dap-ectABC的发酵
将谷氨酸棒杆菌野生菌株MB001以及重组菌株C.glutamicum lysC-P1-298,C.glutamicum lysC-dap,C.glutamicum lysC-dap-ectABC在LB平板上过夜培养。从该新鲜平板上单菌落接种到含有30ml种子培养基的250ml带档板摇瓶中,32℃,200rpm培养12小时。
种子培养基的配方包括(g/L):葡萄糖40,(NH4)2SO4 5.0,K2HPO4 1.5,MgSO4 1.0,MnSO4 0.005,FeSO4 0.005,玉米浆30。C.glutamicum lysC-dap-ectABC培养基中额外添加5mg/L氯霉素。
以10%的接种量将种子液接种到2L发酵培养基当中,发酵采用5L的发酵罐,控制温度为32℃,通气量为1vvm,调整转速使得溶氧水平保持在10%以上,通过流加氨水控制pH值稳定在7.0左右。
发酵培养基配方包括(g/L):葡萄糖80,(NH4)2SO4 30.0,K2HPO4 2.5,MgSO4 1.0,MnSO4 0.010,FeSO4 0.010,玉米浆15,生物素0.0005,盐酸硫胺素0.005。C.glutamicumlysC-dap-ectABC培养基中额外添加5mg/L氯霉素,并在发酵3h时添加1mM的IPTG。
发酵72小时,通过液相色谱检测产物浓度。野生菌株MB001不产氨基酸及四氢嘧啶,实施例1得到的重组菌C.glutamicum lysC-P1-298产18g/L的赖氨酸但不产四氢嘧啶。实施例2得到的重组菌C.glutamicum lysC-dap产6g/L的赖氨酸及2g/L的甘氨酸但不产四氢嘧啶。实施例3得到的重组菌C.glutamicum lysC-dap-ectABC产2g/L的赖氨酸及12g/L的四氢嘧啶。
可见,在本申请实施例1和2的基础上,进一步构建的重组菌C.glutamicum lysC-dap-ectABC能够发酵底物生产四氢嘧啶,与现有技术的其他菌相比,生产四氢嘧啶的量在同一个水平级上,但由于本发明的重组菌谷氨酸棒杆菌C.glutamicum lysC-dap-ectABC为食品安全级,不会产生内毒素,省去了在后续工程去除毒素分离提纯产物的繁琐步骤,不仅提高了产品的安全性,同时极大地降低了生产成本。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 清华大学
<120> 一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法
<130> KHP171112919.2
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 1372
<212> DNA
<213> 突变后的lysC基因及启动子
<400> 1
ggtgcacaaa gcaaaagctg ggtacctcta tctggtgccc taaacggggg aatattaacg 60
ggcccagggt ggtcgcacct tggttggtag gagtagcatg ggatccatgg ccctggtcgt 120
acagaaatat ggcggttcct cgcttgagag tgcggaacgc attagaaacg tcgctgaacg 180
gatcgttgcc accaagaagg ctggaaatga tgtcgtggtt gtctgctccg caatgggaga 240
caccacggat gaacttctag aacttgcagc ggcagtgaat cccgttccgc cagctcgtga 300
aatggatatg ctcctgactg ctggtgagcg tatttctaac gctctcgtcg ccatggctat 360
tgagtccctt ggcgcagaag cccaatcttt cacgggctct caggctggtg tgctcaccac 420
cgagcgccac ggaaacgcac gcattgttga tgtcactcca ggtcgtgtgc gtgaagcact 480
cgatgagggc aagatctgca ttgttgctgg tttccagggt gttaataaag aaacccgcga 540
tgtcaccacg ttgggtcgtg gtggttctga caccactgca gttgcgttgg cagctgcttt 600
gaacgctgat gtgtgtgaga tttactcgga cgttgacggt gtgtataccg ctgacccgcg 660
catcgttcct aatgcacaga agctggaaaa gctcagcttc gaagaaatgc tggaacttgc 720
tgctgttggc tccaagattt tggtgctgcg cagtgttgaa tacgctcgtg cattcaatgt 780
gccacttcgc gtacgctcgt cttatagtaa tgatcccggc actttgattg ccggctctat 840
ggaggatatt cctgtggaag aagcagtcct taccggtgtc gcaaccgaca agtccgaagc 900
caaagtaacc gttctgggta tttccgataa gccaggcgag gctgcgaagg ttttccgtgc 960
gttggctgat gcagaaatca acattgacat ggttctgggc aacgtctctt ctgtagaaga 1020
cggcaccacc gacatcacct tcacctgccc tcgttccgac ggccgccgcg cgatggagat 1080
cttgaagaag cttcaggttc agggcaactg gaccaatgtg ctttacgacg accaggtcgg 1140
caaagtctcc ctcgtgggtg ctggcatgaa gtctcaccca ggtgttaccg cagagttcat 1200
ggaagctctg cgcgatgtca acgtgaacat cgaattgatt tccacctctg agattcgtat 1260
ttccgtgctg atccgtgaag atgatctgga tgctgctgca cgtgcattgc atgagcagtt 1320
ccagctgggc ggcgaagacg aagccgtcgt ttatgcaggc accggacgct aa 1372
<210> 2
<211> 1105
<212> DNA
<213> 突变后的dapA基因及启动子
<400> 2
gcaaagctca cacccacgag ctaaaaattc atatagttaa gacaacattt ttggctgtaa 60
aagacagccg taaaaacctc ttgctcgtgt caattgttct tatcggaatg tggcttgggc 120
gattgttatg caaaagttgt taggtttttt gcggggttgt ttaaccccca aatgagggaa 180
gaaggaaacc ttgaactcta tgagcacagg tttaacagct aagaccggag tagagcactt 240
cggcaccgtt ggagtagcaa tggttactcc attcacggaa tccggagaca tcgatatcgc 300
tgctggccgc gaagtcgcgg cttatttggt tgataagggc ttggattctt tggttctcgc 360
gggcaccact ggtgaatccc caacgacaac cgccgctgaa aaactagaac tgctcaaggc 420
cgttcgtgag gaagttgggg atcgggcgaa gctcatcgcc ggtgtcggaa ccaacaacac 480
gcggacatct gtggaacttg cggaagctgc tgcttctgct ggcgcagacg gccttttagt 540
tgtaactcct tattactcca agccgagcca agagggattg ctggcgcact tcggtgcaat 600
tgctgcagca acagaggttc caatttgtct ctatgacatt cctggtcggt caggtattcc 660
aattgagtct gataccatga gacgcctgag tgaattacct acgattttgg cggtcaagga 720
cgccaagggt gacctcgttg cagccacgtc attgatcaaa gaaacgggac ttgcctggta 780
ttcaggcgat gacccactaa accttgtttg gcttgctttg ggcggatcag gtttcatttc 840
cgtaattgga catgcagccc ccacagcatt acgtgagttg tacacaagct tcgaggaagg 900
cgacctcgtc cgtgcgcggg aaatcaacgc caaactatca ccgctggtag ctgcccaagg 960
tcgcttgggt ggagtcagct tggcaaaagc tgctctgcgt ctgcagggca tcaacgtagg 1020
agatcctcga cttccaatta tggctccaaa tgagcaggaa cttgaggctc tccgagaaga 1080
catgaaaaaa gctggagttc tataa 1105
<210> 3
<211> 2432
<212> DNA
<213> ectABC
<400> 3
atgaacgcaa ccacagagcc ctttacaccc tccgccgacc tggccaagcc cagcgtggcc 60
gatgccgtgg tcggccatga ggcctcaccg ctcttcatcc gcaagccaag ccccgatgac 120
ggctggggca tctacgagct ggtcaagtcc tgtccgcctc tcgacgtcaa ttccgcctac 180
gcctatctgt tgctggccac ccagttccgc gatagctgcg ccgtggcgac caacgaagag 240
ggcgagatcg tcggcttcgt ttccggctac gtgaagagca acgcccccga tacctatttc 300
ctctggcagg ttgccgtggg cgagaaggca cgtggcaccg gcctggcccg tcgtctggtg 360
gaagccgtga tgacacgccc ggaaatggcc gaggtccacc atctcgagac cactatcacg 420
cccgacaacc aggcgtcctg gggcttgttc cgccgtctcg ccgatcgctg gcaggcgccg 480
ttgaacagcc gcgaatactt ctccaccgat caactcggcg gtgagcatga cccggaaaac 540
ctcgttcgca tcggcccgtt ccagaccgac cagatctgag ccgggacgcc gcctggccgg 600
cccggtacgg gccggcaacc cgtcttttcg ttttatcact ttcccccaca ggaggtcgca 660
atgcagaccc agattctcga acgcatggag tccgacgttc ggacctactc ccgctccttc 720
ccggtcgtct tcaccaaggc gcgcaatgcc cgcctgaccg acgaggaagg gcgcgagtac 780
atcgacttcc tggccggtgc cggcaccctg aactacggcc acaacaaccc gcacctcaag 840
caggcgctgc tcgactatat cgacagcgac ggcatcgtcc acggcctgga cttctggact 900
gcggccaagc gcgactatct ggaaaccctg gaagaggtga tcctcaagcc gcgcggtctc 960
gactacaagg tgcatctgcc cggaccgact ggcaccaacg ccgtcgaggc ggccattcgc 1020
ctggcccggg tcgccaaggg gcgccacaat atcgtctcct tcaccaacgg ctttcatggc 1080
gtcaccatgg gcgcgctggc gaccaccggt aaccgcaagt tccgcgaggc caccggtggc 1140
gtgccgaccc aggctgcttc cttcatgccg ttcgatggct acctcggcag cagcaccgac 1200
accctcgact acttcgagaa gctgctcggc gacaagtccg gcggcctgga cgtgcccgcg 1260
gcggtgatcg tcgagacagt gcagggcgag ggcggtatca atgtcgccgg cctggagtgg 1320
ctcaagcgcc tcgagagcat ctgccgcgcc aatgacatcc tgctgatcat cgacgacatc 1380
caggcgggct gcggccggac cggcaagttc ttcagcttcg agcatgccgg catcacgccg 1440
gatatcgtga ccaactccaa gtcgctgtcc ggttacggcc tgccgttcgc tcacgtcctg 1500
atgcgccccg agctcgacaa gtggaagccc ggtcagtaca acggcacctt ccgcggcttc 1560
aacctggctt tcgccactgc tgctgccgcc atgcgcaagt actggagcga cgacaccttc 1620
gagcgtgacg tgcagcgcaa ggctcgcatc gtcgaggaac gcttcggcaa gatcgccgcc 1680
tggctgagcg agaacggcat cgaggcctcc gagcgcggcc gcgggctgat gcggggcatc 1740
gacgtgggtt ccggcgatat cgccgacaag atcacccacc aagccttcga gaacgggttg 1800
atcatcgaaa ccagcggtca ggacggcgaa gtggtcaagt gcctgtgccc gctgaccatt 1860
cccgacgaag acctggtcga gggactcgac atcctcgaga ccagcaccaa gcaggccttt 1920
agctgatcgc ctgaggtgcg ccatcgggcc tgtccatggc atcctgtatc ggtcggccgt 1980
gcgcggccgg ccagtcattg attcactgga gaatcgacat gatcgttcgc aatctcgaag 2040
aagcgcgcca gaccgaccgt ctggtcaccg ccgaaaacgg caactgggac agcacccgcc 2100
tgtcgctggc cgaagatggt ggcaactgct ccttccacat cacccgcatc ttcgagggta 2160
ccgagaccca catccactat aagcatcact tcgaggctgt ttattgcatc gaaggcgagg 2220
gcgaagtgga aaccctggcc gatggcaaga tctggcccat caagccgggt gacatctaca 2280
tcctcgacca gcacgacgag cacctgctgc gcgccagcaa gaccatgcac ctggcctgcg 2340
tgttcacgcc gggcctgacc ggcaacgaag tgcaccgcga agacggttcc tacgcacctg 2400
ccgacgaagc cgacgaccag aagccgctgt aa 2432
<210> 4
<211> 106
<212> DNA
<213> 启动子
<400> 4
ggtgcacaaa gcaaaagctg ggtacctcta tctggtgccc taaacggggg aatattaacg 60
ggcccagggt ggtcgcacct tggttggtag gagtagcatg ggatcc 106
<210> 5
<211> 47
<212> DNA
<213> 人工序列
<400> 5
aggaaacagc tatgacatga ttacgtttcg cccagaacca agtagcc 47
<210> 6
<211> 35
<212> DNA
<213> 人工序列
<400> 6
cccagctttt gctttgtgca cctttcgatc tacgt 35
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<400> 7
catgggatcc atggccctgg tcgtacagaa 30
<210> 8
<211> 39
<212> DNA
<213> 人工序列
<400> 8
gagacgttgc ccagaaccat gtcaatgttg atttctgca 39
<210> 9
<211> 36
<212> DNA
<213> 人工序列
<400> 9
acatggttct gggcaacgtc tcttctgtag aagacg 36
<210> 10
<211> 41
<212> DNA
<213> 人工序列
<400> 10
tgcatgcctg caggtcgact acataaggtc cgacatcgcc t 41
<210> 11
<211> 43
<212> DNA
<213> 人工序列
<400> 11
ctatgacatg attacgaatt cagatggttt tcctgaccag ctt 43
<210> 12
<211> 35
<212> DNA
<213> 人工序列
<400> 12
gggaagaagg aaaccttgaa ctctatgagc acagg 35
<210> 13
<211> 31
<212> DNA
<213> 人工序列
<400> 13
ttcaaggttt ccttcttccc tcatttgggg g 31
<210> 14
<211> 43
<212> DNA
<213> 人工序列
<400> 14
tgcctgcagg tcgactctag aggcctgtaa aggctcattt cag 43
<210> 15
<211> 41
<212> DNA
<213> 人工序列
<400> 15
tgcatgcctg caggtcgact aggaggccct tcagatgaac g 41
<210> 16
<211> 40
<212> DNA
<213> 人工序列
<400> 16
ccgccaaaac agccaagctg ttacagcggc ttctggtcgt 40
<210> 17
<211> 36
<212> DNA
<213> 人工序列
<400> 17
ggtgcacaaa gcaaaagctg ggtacctcta tctggt 36
<210> 18
<211> 28
<212> DNA
<213> 人工序列
<400> 18
ccagggccat ggatcccatg ctactcct 28
Claims (10)
1.一种重组谷氨酸棒杆菌,其特征在于,含有突变后的lysC基因及启动子,突变后的dapA基因及启动子和ectABC基因;
所述突变后的lysC基因及启动子的序列如SEQ ID NO.1所示;
所述突变后的dapA基因及启动子的序列如SEQ ID NO.2所示;
所述ectABC基因的序列如SEQ ID NO.3所示。
2.如权利要求1所述的重组谷氨酸棒杆菌,其特征在于,通过以下方法制备得到:
(1)将解除反馈抑制的天冬氨酸激酶基因lysC和强启动子片段P1连接到自杀性质粒上,获得的重组质粒,将该重组质粒电转化入谷氨酸棒杆菌中,得到重组菌C.glutamicumlysC-P1-298;
(2)通过启动子置换弱化重组菌C.glutamicum lysC-P1-298中的二氢嘧啶二羧酸合成酶基因dapA的表达,得到重组菌C.glutamicum lysC-dap;
(3)在重组菌C.glutamicum lysC-dap中转入能够过表达四氢嘧啶合成途径相关基因ectABC的表达载体,构建得到重组菌C.glutamicum lysC-dap-ectABC。
3.如权利要求2所述的重组谷氨酸棒杆菌,其特征在于,步骤(1)是将lysC1,lysC2,lysC3和强启动子片段P1连接到自杀性质粒上,所述lysC1,lysC2,lysC3分别通过引物对SEQ ID NO.5-6,SEQ ID NO.7-8,SEQ ID NO.9-10以谷氨酸棒杆菌基因组为模板PCR扩增得到。
4.如权利要求2所述的重组谷氨酸棒杆菌,其特征在于,所述强启动子片段P1,其序列如SEQ ID NO.4所示,所述自杀性质粒为pK18mobsacB。
5.如权利要求2所述的重组谷氨酸棒杆菌,其特征在于,步骤(2)是将dap1和dap2片段连接到自杀性质粒上,获得的重组质粒;
所述dap1和dap2片段分别通过引物对SEQ ID NO.11-12,SEQ ID NO.13-14以谷氨酸棒杆菌基因组为模板PCR扩增得到。
6.如权利要求2-5任一所述的重组谷氨酸棒杆菌,其特征在于,步骤(3)是将ectABC基因连接到表达载体pXMJ19上,获得的重组质粒命名为pXMJ-ectABC,将该重组质粒电转化入步骤(2)的重组菌C.glutamicum lysC-dap中;所述ectABC基因通过引物对SEQ ID NO.15-16以Halomonas elongata DSM 2581基因组为模板PCR扩增得到。
7.权利要求1-6任一所述的重组谷氨酸棒杆菌的培养基,其特征在于,含有氯霉素。
8.利用权利要求1-6任一所述的重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法,其特征在于,以含可发酵糖的原料为底物,接入权利要求1-6任一所述的重组谷氨酸棒杆菌,发酵温度28-40℃,pH值5-8,溶氧值10%进行发酵。
9.如权利要求8所述的方法,其特征在于,所述含可发酵糖的原料为糖蜜,蔗糖、葡萄糖、淀粉水解液、玉米浆、木糖、甘露糖或甘油;发酵温度为32-35℃,pH值6-7,在发酵3-5h时添加终浓度为0.01-1mM的IPTG。
10.如权利要求8或9所述的方法,其特征在于,发酵过程中流加碳源,以及发酵培养基中含有硫胺素和生物素。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101698845A (zh) * | 2009-06-29 | 2010-04-28 | 中国科学院微生物研究所 | 源自谷氨酸棒杆菌的启动子及其应用 |
CN105018403A (zh) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | 一种产生四氢嘧啶的基因工程菌及其构建方法与应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101250548B (zh) | 2007-03-30 | 2010-06-02 | 中国科学院微生物研究所 | 一种穿梭质粒及其衍生质粒 |
EP2374873A1 (en) | 2010-04-08 | 2011-10-12 | Technische Universität Hamburg-Harburg | Modified aspartate kinase from corynebacterium and its application for amino acid production |
CN107142234B (zh) | 2017-05-12 | 2020-09-15 | 清华大学 | 一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法 |
-
2017
- 2017-05-12 CN CN201710336026.7A patent/CN107142234B/zh active Active
- 2017-11-30 US US16/612,692 patent/US11512333B2/en active Active
- 2017-11-30 WO PCT/CN2017/113806 patent/WO2018205563A1/zh active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101698845A (zh) * | 2009-06-29 | 2010-04-28 | 中国科学院微生物研究所 | 源自谷氨酸棒杆菌的启动子及其应用 |
CN105018403A (zh) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | 一种产生四氢嘧啶的基因工程菌及其构建方法与应用 |
Non-Patent Citations (4)
Title |
---|
CHEN Z ET AL: "Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Cornebacterium glutamicum", 《APPL ENVIRON MICROBIOL》 * |
F PEREZ GARCIA ET AL: "Improved fermentative production of the compatible solute ectoine by cornebacterium glutamicum from glucose and alternative carbon sources", 《JOURNAL OF BIOTECHNOLOGY》 * |
J BECKER ER AL: "Systems metabolic engineering of corynebacterium glutamicum for production of the chemical chaperone ectoine", 《MICROB CELL FACT》 * |
ZHEN CHEN ET AL: "Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in corynebacterium glutamicum for lysine production", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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WO2018205563A1 (zh) * | 2017-05-12 | 2018-11-15 | 清华大学 | 一种利用重组谷氨酸棒杆菌发酵生产四氢嘧啶的方法 |
US11512333B2 (en) | 2017-05-12 | 2022-11-29 | Beijing KansenBio Technology Ltd. | Method for producing tetrahydropyrimidine by fermenting recombinant Corynebacterium glutamicum |
CN111394288A (zh) * | 2019-01-03 | 2020-07-10 | 北京百奥茵诺生物科技有限公司 | 重组谷氨酸棒杆菌、其构建方法及其生产四氢嘧啶的方法 |
CN111394288B (zh) * | 2019-01-03 | 2022-05-03 | 北京百奥茵诺生物科技有限公司 | 重组谷氨酸棒杆菌、其构建方法及其生产四氢嘧啶的方法 |
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CN111893146A (zh) * | 2020-08-26 | 2020-11-06 | 无锡晶扬生物科技有限公司 | 提高谷氨酸棒杆菌四氢嘧啶产量的培养基及发酵方法 |
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CN114540261B (zh) * | 2020-11-24 | 2024-02-02 | 北京化工大学 | 一种产氨基己二酸的基因工程菌 |
WO2023142848A1 (zh) * | 2022-01-26 | 2023-08-03 | 廊坊梅花生物技术开发有限公司 | 启动子、产苏氨酸重组微生物及其应用 |
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WO2018205563A1 (zh) | 2018-11-15 |
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