CN115011620A - 一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成l-苏氨酸的方法 - Google Patents
一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成l-苏氨酸的方法 Download PDFInfo
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- CN115011620A CN115011620A CN202210552528.4A CN202210552528A CN115011620A CN 115011620 A CN115011620 A CN 115011620A CN 202210552528 A CN202210552528 A CN 202210552528A CN 115011620 A CN115011620 A CN 115011620A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成L‑苏氨酸的方法,涉及生物工程技术领域。本发明所述大肠杆菌重组核酸,利用磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因对大肠杆菌进行改造,获得一株以葡萄糖为底物的重组大肠杆菌LMT4,利用所述LMT4进行发酵生产,L‑苏氨酸产量以及转化率明显提高,为L‑苏氨酸的工业化生产奠定基础。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成L-苏氨酸的方法。
背景技术
苏氨酸是一种必需的氨基酸,主要用于医药、化学试剂、食品强化剂、饲料添加剂等方面。由于苏氨酸的结构中含有羟基,对人体皮肤具有持水作用,与寡糖链结合,对保护细胞膜起重要作用,在体内能促进磷脂合成和脂肪酸氧化。苏氨酸制剂具有促进人体发育和抗脂肪肝的药用效能,是复合氨基酸输液中的一个成分。同时苏氨酸是猪饲料的第二限制氨基酸和家禽饲料的第三限制氨基酸,近年来全球人口对肉类物品的需求呈现持续快速增长的趋势,因此L-苏氨酸的市场需求也在不断增加。
目前,L-苏氨酸生产方法主要有化学合成法、蛋白水解法和微生物发酵法,其中微生物发酵法生产成本低、生产强度高、对环境污染小,成为工业化生产L-苏氨酸最广泛的方法。目前生产L-苏氨酸的菌株主要包括大肠杆菌、谷氨酸棒杆菌、粘质沙雷氏菌。SangYupLee等运用系统生物学方法从E coli W3100(1acI-)出发,构建的工程菌株发酵50h产L-苏氨酸82.4g/L,糖酸转化率为39.3%。天津大学乔建军以THRD为出发菌株,通过两段碳分配和辅因子生成策略提高L-苏氨酸的产量,菌株40h生成70.8g/L的L-苏氨酸,糖酸转化率40.4%。华东理工大学沈琼通过强化L-苏氨酸合成途径关键酶和L-苏氨酸分泌有关基因,构建基因工程菌E coli VNBKB.3507,其发酵48h产L-苏氨酸52.7g/L。江南大学王小元通过代谢工程改造大肠杆菌TWF001,经过36h摇瓶培养后,可产生15.85g/L L-苏氨酸,糖酸转化率为53%。
目前,利用大肠杆菌生产苏氨酸的菌株,L-苏氨酸的产量和糖酸转化率仍然有很大的提升空间,而且通过质粒过表达关键基因的方法需要在发酵过程中加入抗生素来保持质粒的稳定性,不仅增加了安全隐患也提高了发酵成本,因此开发一种无质粒的高效合成L-苏氨酸重组菌株,对工业生产具有重要意义。
发明内容
有鉴于此,本发明的目的在于提供一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成L-苏氨酸的方法,基于系统代谢工程改造和优化策略,获得高效合成L-苏氨酸的大肠杆菌重组菌株。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种大肠杆菌重组核酸,所述重组核酸包括磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因。
优选的,所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因均由Trc启动子启动表达。
优选的,所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因来源于枯草芽孢杆菌;
所述丙酮酸羧化酶pyc的编码基因来源于地衣芽孢杆菌。
优选的,所述磷酸烯醇式丙酮酸羧化激酶pck经RBS优化和143位甘氨酸突变为精氨酸;
所述丙酮酸羧化酶pyc经RBS优化和247位丙氨酸突变为赖氨酸。
优选的,所述苏氨酸操纵子经RBS优化和144位丙氨酸突变为天冬氨酸。
本发明还提供了一种包含上述重组核酸的重组大肠杆菌,所述重组大肠杆菌过表达磷酸烯醇式丙酮酸羧化激酶pck、丙酮酸羧化酶pyc和苏氨酸操纵子。
优选的,所述重组大肠杆菌的基础菌株包括大肠杆菌K-12W3110。
本发明还提供了上述重组大肠杆菌的培养方法,包括以下步骤:将所述重组大肠杆菌接种于种子培养基上进行培养,得种子液;所述种子培养基包括以下浓度的组分:玉米浆干粉5g/L,葡萄糖20g/L,酵母粉5g/L,KH2PO4 2g/L,硫酸镁1g/L,FeSO4·7H2O 20mg/L和MnSO4·H2O 20mg/L。
本发明还提供了上述重组大肠杆菌在生物合成L-苏氨酸中的应用,所述重组大肠杆菌以葡萄糖为发酵底物。
本发明还提供了一种生物合成L-苏氨酸的方法,包括以下步骤:将利用上述培养方法得到的种子液接种于发酵培养基中,进行有氧发酵,发酵液中含L-苏氨酸;
所述发酵培养基包括以下浓度的组分:葡萄糖20g/L,磷酸二氢钾2g/L,酵母粉3g/L,甜菜碱1g/L,硫酸镁1g/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,玉米浆干粉8g/L和维生素B110mg/L。
有益效果:本发明提供了大肠杆菌重组核酸,包括磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因。利用所述重组核酸对大肠杆菌的基因组进行改造,利用CRISPR-Cas9技术对质粒进行基因改造,通过CRISPR-Cas9技术完成基因改造目的后,会将CRISPR-Cas9体系中的质粒从大肠杆菌中去除,因此在最后苏氨酸发酵过程中获得不含质粒的重组大肠杆菌LMT4,以葡萄糖为底物,利用所述重组大肠杆菌LMT4进行发酵生产,L-苏氨酸产量以及转化率明显提高,为L-苏氨酸的工业化生产奠定基础。本发明实施例中,利用所述重组大肠杆菌LMT4,以葡萄糖为底物,在5L发酵罐内发酵48h,可产生160g/L苏氨酸,糖酸转化率达到60%,表明本发明所述高效合成L-苏氨酸的重组菌株具有广泛的工业应用前景。
附图说明
图1为重组菌株LMT4在5L发酵罐中L-苏氨酸产量图;
图2为重组菌株LMT4在5L发酵罐中生长曲线图;
图3为pGRB载体质粒图;
图4为CRISPR-Cas9质粒图谱。
具体实施方式
本发明提供了一种大肠杆菌重组核酸,所述重组核酸包括磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因。
本发明优选以大肠杆菌为出发菌株,并通过基因编辑的方式,敲除所述出发菌株的假基因yeeL并在yeeL位点整合所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因;敲除所述出发菌株的假基因yjhE并在yjhE位点整合丙酮酸羧化酶pyc的编码基因;和敲除所述出发菌株的假基因ydeu并在ydeu位点整合苏氨酸操纵子的编码基因。在本发明中,所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因优选均由Trc启动子启动表达。本发明对所述基因编辑的方法并没有特殊限定,优选包括CRISPR Cas9。
本发明所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因优选来源于枯草芽孢杆菌,在整合进yeeL位点前优选还包括对所述磷酸烯醇式丙酮酸羧化激酶pck进行RBS优化和143位甘氨酸突变为精氨酸。本发明所述RBS优化优选为将位于磷酸烯醇式丙酮酸羧化激酶pck上游的用于调控磷酸烯醇式丙酮酸羧化激酶pck的RBS序列替换为SEQ ID NO.2:CATCAGATAGGTGTAAGGAGGTTTAGAT。经本发明所述RBS优化和突变后,整合进yeeL位点的磷酸烯醇式丙酮酸羧化激酶pck的完整编码基因序列优选如SEQ ID NO.1所示。
本发明所述丙酮酸羧化酶pyc的编码基因优选来源于地衣芽孢杆菌,且在整合进yjhE位点前,优选还包括对所述丙酮酸羧化酶pyc进行RBS优化和247位丙氨酸突变为赖氨酸,所述RBS优化优选为将位于丙酮酸羧化酶pyc上游的用于调控丙酮酸羧化酶pyc的RBS序列进行替换,所述替换后的RBS序列的核苷酸序列优选如SEQ ID NO.4所示:CAACAGATAGGTGTAAGGAGGTTGAGAT。经本发明所述RBS优化和突变后,整合进yjhE位点的丙酮酸羧化酶pyc的完整编码基因序列优选如SEQ ID NO.3所示。
本发明所述苏氨酸操纵子的144位丙氨酸突变为天冬氨酸(thrABA144DC),经所述突变后,thrABA144DC的编码基因的核苷酸序列优选如SEQ ID NO.5所示:TTTCACACAGGAAACAGA;同时对SEQ ID NO.5所示的序列进行RBS优化,所述RBS优化优选为将SEQ ID NO.5所示序列中的RBS序列替换为SEQ ID NO.6所示的序列:CGGTAAAGATATCGATAAGGAGGTTTTTT,而后将经所述突变和RBS优化后的thrABA144DC整合进ydeu位点。
本发明还提供了一种包含上述重组核酸的重组大肠杆菌,所述重组大肠杆菌过表达磷酸烯醇式丙酮酸羧化激酶pck、丙酮酸羧化酶pyc和苏氨酸操纵子。
本发明所述重组大肠杆菌的基础菌株优选包括大肠杆菌K-12W3110,所述基础菌株缺失了DNA结合转录抑制因子LacI、苏氨酸前导肽编码基因thrL、苏氨酸钠离子转运体基因sstT、苏氨酸脱氢酶tdh、苏氨酸转运蛋白tdcC。本发明所述大肠杆菌W3110优选购自北纳生物。
本发明还提供了所述重组大肠杆菌的构建方法,优选利用CRISPR Cas9(图4)的方法进行构建,更优选包括:(1)从大肠杆菌K-12W3110的基因组上PCR扩增假基因yeeL的上游同源臂和下游同源臂;(2)利用表1中引物pck-1、pck-2、pck-3、pck-4,从枯草芽孢杆菌基因组中扩增pckG143R基因,得到片段1-PCK、2-PCK,再以pck-1、pck-4为引物,片段1-pckG143R、2-pckG143R为模板,将片段1-pckG143R、2-pckG143R融合为pckG143R,其中pck-1引物中包含优化后的RBS序列,pck-2和pck-3为pckG143R基因点突变引物,通过PCR完成磷酸烯醇式丙酮酸羧化激酶pckG143R基因RBS的优化和点突变;(3)将yeeL的上、下游同源臂和Trc启动子驱动的磷酸烯醇式丙酮酸羧化激酶pckG143R片段融合,得到U-pckG143R-D片段;(4)将得到的融合片段U-pckG143R-D和含有yeel-sgRNA的载体转化进重组大肠杆菌K-12W3110(简称LMT1),得到敲除了假基因yeeL并在yeeL位点整合由Trc启动子驱动的枯草芽孢杆菌来源的磷酸烯醇式丙酮酸羧化激酶pckG143R的重组菌株,去除yeel-sgRNA载体后得到重组菌株LMT2;
(5)从大肠杆菌K-12W3110的基因组上PCR扩增假基因yjhE的上游同源臂和下游同源臂;(6)利用表1中引物pyc-1、pyc-2和pyc-3、pyc-4,从地衣芽孢杆菌基因组中扩增pycA247K基因,得到片段1-pycA247K、2-pycA247K,再以pyc-1、pyc-4为引物,片段1-pycA247K、2-pycA247K为模板,将片段1-pycA247K、2-pycA247K融合为pycA247K,其中pyc-1引物中包含优化后的RBS序列,pyc-2和pyc-3为pycA247K基因点突变引物,通过PCR完成丙酮酸羧化酶pycA247K基因RBS的优化和点突变;(7)将yjhE的上、下游同源臂和Trc启动子驱动的丙酮酸羧化酶pycA247K片段融合,得到U-pyc-D片段;(8)将得到的融合片段U-pycA247K-D和含有yjhE-sgRNA的载体转化进重组菌株LMT2,得到敲除了假基因yjhE并在yjhE位点整合由Trc启动子驱动的丙酮酸羧化酶pycA247K的重组菌株,去除yjhE-sgRNA载体后得到重组菌株LMT3;
(9)从大肠杆菌K-12W3110的基因组上PCR扩增假基因ydeu的上游同源臂和下游同源臂;(10)利用表1中引物thrA-F、thrB-R和thrB-F、thrC-R,从大肠杆菌K-12W3110基因组上PCR扩增Trc启动子驱动的苏氨酸操纵子thrABA144DC基因簇,得到片段1-thrABA144DC、2-thrABA144DC,再以thrA-F、thrC-R为引物,片段1-thrABA144DC、2-thrABA144DC为模板,将片段1-thrABA144DC、2-thrABA144DC融合为thrABA144DC,其中thrA-F引物中包含优化后的RBS序列,thrB-R和thrB-F为thrABA144DC基因点突变引物,通过PCR完成苏氨酸操纵子thrABA144DC基因RBS的优化和点突变;(11)将ydeu的上、下游同源臂和Trc启动子驱动的苏氨酸操纵子thrABA144DC基因簇片段融合,得到U-thrABA144DC-D片段;(12)将得到的融合片段U-thrABA144DC-D和含有ydeu-sgRNA的载体转化进重组菌株LMT3,得到敲除了假基因ydeu并在ydeu位点整合由Trc启动子驱动的苏氨酸操纵子thrABA144DC基因簇的重组菌株,去除ydeu-sgRNA载体后得到重组大肠杆菌菌株LMT4。
在本发明中,为完成所述重组大肠杆菌的构建,利用了表1所示的引物。
表1引物信息
本发明还提供了上述重组大肠杆菌的培养方法,包括以下步骤:将所述重组大肠杆菌接种于种子培养基上进行培养,得种子液;所述种子培养基包括以下浓度的组分:玉米浆干粉5g/L,葡萄糖20g/L,酵母粉5g/L,KH2PO42g/L,硫酸镁1g/L,FeSO4·7H2O 20mg/L和MnSO4·H2O 20mg/L。
本发明所述接种的接种量优选为20%。本发明所述培养的温度优选为37℃,且在所述培养时伴随震荡,所述震荡的频率优选为500rpm,所述培养的时间优选为10h。经本发明所述培养后,OD600:12~15。
本发明所述重组大肠杆菌可以以葡萄糖为底物,生物合成L-苏氨酸,L-苏氨酸产量以及转化率明显提高,为L-苏氨酸的工业化生产奠定基础。
本发明还提供了上述重组大肠杆菌在生物合成L-苏氨酸中的应用。
通过本发明改造的大肠杆菌W3110具有高产性能:1、过表达枯草芽孢杆菌来源磷酸烯醇式丙酮酸羧化酶pck可以催化磷酸烯醇式丙酮酸生成草酰乙酸,而草酰乙酸是苏氨酸的前体物质。同时在磷酸烯醇式丙酮酸羧化酶pck的催化过程中还会生成能量ATP,苏氨酸合成过程中会消耗ATP,所以该技术既可以提高苏氨酸合成前体物的供应,也可以提供合成过程中所需要的能量ATP,通过RBS优化策略提高了磷酸烯醇式丙酮酸羧化酶在大肠杆菌内的表达,点突变提高了枯草芽孢杆菌来源磷酸烯醇式丙酮酸羧化酶的热稳定性和催化效率,使磷酸烯醇式丙酮酸转化为草酰乙酸的速率更快。2、过表达地衣芽孢杆菌来源的丙酮酸羧化酶,可以催化丙酮酸合成草酰乙酸,而大肠杆菌中是没有丙酮酸羧化酶存在的,所以通过异源表达丙酮酸羧化酶拓展了大肠杆菌内丙酮酸合成草酰乙酸的代谢途径,增加了苏氨酸前体物的积累。通过RBS优化策略提高了式丙酮酸羧化酶在大肠杆菌内的表达,点突变提高了地衣芽孢杆菌来源丙酮酸羧化酶的热稳定性和催化效率,使丙酮酸转化为草酰乙酸的速率更快。3、过表达大肠杆菌内源性苏氨酸操纵子thrABA144DC,增加了苏氨酸合成方向的代谢通量,通过RBS优化进一步提高了酶的表达水平,点突变提高了thrB的催化效率。通过上述发明专利可以使菌株合成L-苏氨酸的能力提高,同时这些过表达的基因都是整合到大肠杆菌基因组上不是通过质粒过表达,所以不需要在发酵过程中添加抗生素维持质粒的存在。
本发明还提供了一种生物合成L-苏氨酸的方法,包括以下步骤:将利用上述培养方法得到的种子液接种于发酵培养基中,进行有氧发酵,发酵液中含L-苏氨酸;
所述发酵培养基包括以下浓度的组分:葡萄糖20g/L,磷酸二氢钾2g/L,酵母粉3g/L,甜菜碱1g/L,硫酸镁1g/L,FeSO4·7H2O 10mg/L,MnSO4·H2O10mg/L,玉米浆干粉8g/L和维生素B110mg/L。
本发明所述种子液的接种体积优选为所述发酵培养基体积的20%,经所述接种后进行有氧发酵,所述有氧发酵的温度为37℃,溶氧30%,在所述有氧发酵过程中,在底糖耗尽后,通过流加葡萄糖将残糖控制在0~1g/L。
下面结合实施例对本发明提供的一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成L-苏氨酸的方法进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、融合片段U-pck-D的构建
利用表1中引物yeeL-U-F、yeeL-U-R、yeeL-D-F和yeeL-D-R,从大肠杆菌K-12W3110基因组中分别扩增yeeL基因两侧上、下游同源臂片段,得到片段yeel1(SEQ ID NO.8)和yeel2(SEQ ID NO.9),以Escherichia coli W3110总DNA为模板,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,60s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×PhantaMax MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
利用表1中引物pck-1、pck-2和pck-3、pck-4,从枯草芽孢杆菌基因组中扩增pckG143R基因,得到片段1-pckG143R、2-pckG143R,再以pck-1、pck-4为引物,片段1-pckG143R、2-pckG143R为模板,将片段1-pckG143R、2-pckG143R融合为pckG143R,其中pck-1引物中包含优化后的RBS序列,pck-2和pck-3为pck基因点突变引物,通过PCR完成pckG143R基因RBS的优化和点突变,以枯草芽孢杆菌总DNA为模板,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×PhantaMaxMasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
将片段yeel1、pckG143R、yeel2进行融合PCR,得到融合片段U-pckG143R-D(SEQ IDNO.10)以yeel1、yeel2、pckG143R为模板,利用上述引物yeeL-U-F、yeeL-D-R做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×PhantaMaxMasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用。
2、yeel-sgRNA重组质粒的构建
利用引物pGRB-F和pGRB-R,从载体pGRB(图3)上PCR得到线性化载体L-pGRB,利用上述引物pGRB-F和pGRB-R做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用。将设计的sgRNA(sgRNA-yeel-1和sgRNA-yeel-2、sgRNA-ydeU-1和sgRNA-ydeU-2、sgRNA-yjhE-1和sgRNA-yjhE-2)与线性化载体L-pGRB连接构建重组质粒yeel-sgRNA。
3、重组大肠杆菌LMT2的构建
将重组质粒yeel-sgRNA和融合片段U-pckG143R-D转化进大肠杆菌K-12W3110(LMT1),选用引物yeeL-U-F与yeeL-D-R进行菌落PCR筛选转化子,确认融合片段U-pckG143R-D成功整合到yeel位点,加2mM阿拉伯糖在30℃下培养12h,去除重组质粒yeel-sgRNA,得到重组菌株LMT2。
去除质粒的方法:pREDCas9质粒是奇霉素抗性,pGRB质粒是氨苄抗生素抗性。1、将改造成功含有奇霉素和氨苄抗生素两个抗性的菌株接种于10ml LB培养基中,培养基中添加2mM阿拉伯糖和1mM奇霉素,30℃培养12h,取2微升菌液在奇霉素抗性LB平板上划线,30℃培养12h,挑取单菌落分别在奇霉素抗性和氨苄抗生素抗性的LB平板上对点板,30℃培养12h,选取奇霉素抗性平板正常生长而氨苄抗生素抗性平板不长的菌落,为去除重组质粒pGRB的菌株。2、将去除重组质粒pGRB的菌株接种于10ml LB培养基,42℃培养12h,取2微升菌株在无抗生素平板上划线,37℃培养12h,挑取单菌落在奇霉素抗性平板和无抗生素抗性平板上对点板,37℃培养12h,选取无抗生素平板生长,奇霉素抗性平板不生长的菌株,为去除质粒pREDCas9的菌株。
4、融合片段U-pyc-D的构建
利用表1中引物yjhE-U-F、yjhE-U-R、yjhE-D-F和yjhE-D-R,从大肠杆菌K-12W3110基因组中分别扩增yjhE基因两侧上、下游同源臂片段,得到片段yjhE 1(SEQ ID NO.11)和yjhE 2(SEQ ID NO.12),利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,60s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max Master Mix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
利用表1中引物pyc-1、pyc-2和pyc-3、pyc-4,从地衣芽孢杆菌基因组中扩增pycA247K基因,得到片段pycA247K,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
将片段yjhE 1、pycA247K、yjhE 2进行融合PCR,得到融合片段U-pycA247K-D(SEQ IDNO.13),利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用。
5、yjhE-sgRNA重组质粒的构建
根据载体PGRB的序列信息,设计引物PGRB-F、PGRB-R,使用上述引物从载体PGRB上PCR得到线性化载体L-PGRB,将设计的sgRNA与线性化载体L-PGRB连接构建重组质粒yjhE-sgRNA,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×PhantaMax MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;。
6、重组大肠杆菌LMT3的构建
将重组质粒yjhE-sgRNA和融合片段U-pycA247K-D转化进重组菌株LMT2,选用引物yjhE-U-F与yjhE-D-R进行菌落PCR筛选转化子,确认融合片段U-pyc-D成功整合到yjhE位点,加2mM阿拉伯糖在30℃下培养12h,去除重组质粒yjhE-sgRNA,得到重组菌株LMT3。
7、融合片段U-thrABA144DC-D的构建
利用表1中引物ydeu-U-F、ydeu-U-R、ydeu-D-F和ydeu-D-R,从大肠杆菌K-12W3110基因组中分别扩增ydeu基因两侧上、下游同源臂片段,得到片段ydeu 1(SEQ ID NO.14)和ydeu 2(SEQ ID NO.15),利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max Master Mix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
利用表1中引物thrA-F、thrB-R、thrB-F和thrC-R,从大肠杆菌基因组中扩增thrABA144DC基因,得到片段thrABA144DC,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用;
将片段ydeu 1、thrABA144DC和ydeu 2进行融合PCR,得到融合片段U-thrABA144DC-D(SEQ ID NO.16),利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×Phanta Max MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用。
8、ydeu-sgRNA重组质粒的构建
根据载体PGRB的序列信息,设计引物PGRB-F、PGRB-R,使用上述引物从载体PGRB上PCR得到线性化载体L-PGRB,将设计的sgRNA与线性化载体L-PGRB连接构建重组质粒ydeu-sgRNA,利用上述引物做PCR扩增,扩增条件为:95℃预变性,5min;98℃变性,10s,55℃退火,15s,72℃延伸,90s,30个循环;72℃终延伸5min。PCR扩增体系:模板1μL,上下游引物各2μL,灭菌的双蒸馏水20μL,2×PhantaMax MasterMix 25μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收,电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中,-20℃冰箱保存备用。
9、重组大肠杆菌LMT4的构建
将重组质粒ydeu-sgRNA和融合片段U-thrABA144DC-D转化进大肠杆菌LMT3,选用引物ydeu-U-F与ydeu-D-R进行菌落PCR筛选转化子,确认融合片段U-thrABA144DC-D成功整合到ydeu位点,加2mM阿拉伯糖在30℃下培养12h,去除重组质粒ydeu-sgRNA,得到重组菌株LMT4。
实施例2
将实施例1构建得到的重组菌株LMT4接种于种子培养基进行种子培养,然后将种子培养物按照20%的接种量转入发酵培养基培养。
1、5L种子罐工艺控制
a.调温度37℃,pH 7.0,转速500rpm,风量0.3m3/h,全过程温度控制37℃,罐压0.05~0.08MPa,培养周期10h;
b.移种标准:OD600:12-15。
c.种子培养基为玉米浆干粉5g/L,葡萄糖20g/L,酵母粉5g/L,KH2PO42g/L,硫酸镁1g/L,FeSO4·7H2O 20mg/L,MnSO4·H2O 20mg/L;
2、5L发酵罐发酵工艺控制
a.调温度37℃,pH7.0,初始转速300rpm,风量0.3m3/h,全过程温度控制37℃,罐压0.05~0.08MPa;
b.残糖控制:流加葡萄糖将残糖控制在0~1g/L之内;
c.DO控制:0h时,风量0.3m3/h,300rpm,罐压0.05MPa;
d.当DO降到30%以下,通过调整通气量和搅拌转速将溶氧水平控制在30%,至发酵结束;
e.发酵培养基为葡萄糖20g/L,磷酸二氢钾2g/L,酵母粉3g/L,甜菜碱1g/L,硫酸镁1g/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,玉米浆干粉8g/L,维生素B110mg/L。
3、L-苏氨酸的测定方法:
1)样品处理:取发酵48h后的发酵液1mL,离心转速12000rpm,离心10min去除菌体取上清。用去离子水将上清液适当稀释然后经孔径为0.22μm的滤膜过滤。
2)分析方法:OPA柱前衍生
3)色谱条件:
(1)色谱柱:色谱柱C18(250×4.6)mm
(2)柱温:40℃
(3)流动相A:称取3.01g无水乙酸钠于烧杯中,加超纯水溶解并定容至1L,然后加入200μL三乙胺,用5%的醋酸将pH调到7.20±0.05;抽滤后加入5mL四氢呋喃,混合后用0.22μm的无机滤膜抽滤,再放入超声清洗锅中排气20min,备用。
流动相B:称取3.01g无水乙酸钠于烧杯中;加超纯水溶解并定容至200mL;用5%醋酸将pH调到7.20±0.05;再向此溶液加入400mL乙腈和400mL甲醇,混合后抽滤,在放入超声清洗锅中排气20min,备用。
(4)流速:1.0ml/min;
(5)紫外检测器:338nm;
(6)柱温:40℃。
经统计,在5L发酵罐内发酵48h,可产生160g/L苏氨酸,糖酸转化率达到60%。
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> 江南大学
<120> 一种大肠杆菌重组核酸、重组大肠杆菌及培养方法和生物合成 L-苏氨酸的方法
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1584
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
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cataatttat cagcagcaca gctaacagaa aaagtcctct cccgaaacga aggcatttta 120
acatccacag gtgctgttcg cgcgacaaca ggcgcttaca caggacgctc acctaaagat 180
aaattcatcg tggaggaaga aagcacgaaa aataagatcg attggggccc ggtgaatcag 240
ccgatttcag aagaagcgtt tgagcggctg tacacgaaag ttgtcagcta tttaaaggag 300
cgagatgaac tgtttgtttt cgaaggattt gccggagcag acgagaaata caggctgccg 360
atcactgtcg taaatgagtt cgcatggcac aatttatttg cgcggcagct gtttatccgt 420
ccggaaagaa atgataagaa aacagttgag cagccgttca ccattctttc tgctccgcat 480
ttcaaagcgg atccaaaaac agacggcact cattccgaaa cgtttattat tgtctctttc 540
gaaaagcgga caattttaat cggcggaact gagtatgccg gtgaaatgaa gaagtccatt 600
ttctccatta tgaatttcct gctgcctgaa agagatattt tatctatgca ctgctccgcc 660
aatgtcggtg aaaaaggcga tgtcgccctt ttcttcggac tgtcaggaac aggaaagacc 720
accctgtcgg cagatgctga ccgcaagctg atcggtgacg atgaacatgg ctggtctgat 780
acaggcgtct ttaatattga aggcggatgc tacgctaagt gtattcattt aagcgaggaa 840
aaggagccgc aaatctttaa cgcgatccgc ttcgggtctg ttctcgaaaa tgtcgttgtg 900
gatgaagata cacgcgaagc caattatgat gattccttct atactgaaaa cacgcgggca 960
gcttacccga ttcatatgat taataacatc gtgactccaa gcatggccgg ccatccgtca 1020
gccattgtat ttttgacggc tgatgccttc ggagtcctgc cgccgatcag caaactaacg 1080
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cgtggtgtca cgtctcctga aacgacgttt tctacatgct tcggctcacc gttcctgccg 1200
cttcctgctc acgtctatgc tgaaatgctc ggcaaaaaga tcgatgaaca cggcgcagac 1260
gttttcttag tcaataccgg atggaccggg ggcggctacg gcacaggcga acgaatgaag 1320
ctttcttaca ctagagcaat ggtcaaagca gcgattgaag gcaaattaga ggatgctgaa 1380
atgataactg acgatatttt cggcctgcac attccggccc atgttcctgg cgttcctgat 1440
catatccttc agcctgaaaa cacgtggacc aacaaggaag aatacaaaga aaaagcagtc 1500
taccttgcaa atgaattcaa agagaacttt aaaaagttcg cacataccga tgccatcgcc 1560
caggcaggcg gccctctcgt ataa 1584
<210> 2
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catcagatag gtgtaaggag gtttagat 28
<210> 3
<211> 3444
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtcacaac agtctattca aaaagttctt gttgcaaaca gaggggaaat cgctatccgc 60
gtatttcggg cctgcacaga actgaatatc cgtacggtag cgatctattc taaagaagac 120
agcggatctt accacagata caaagccgat gaagcatacc tggtcggcga agggaaaaag 180
ccgattgacg cttatcttga tattgaaggc atcatcgaga ttgcaaaacg caaccatgtg 240
gatgccatcc atccaggcta cggcttcctg tcggaaaaca ttcagtttgc taagcggtgt 300
gaagaggaag gcatcatctt tatcggaccg acctccgagc acctcgatat gtttggagac 360
aaagtaaaag cccgcgaaca agctgaaaaa gctggaattc cggtcatacc ggggagcgac 420
ggaccagtgg cggatatagc ggaagtgaaa caatttgcgg aaaagttcgg atatccgttt 480
atcattaaag cgtcgcttgg cggcggcggg cgcggcatgc ggatcgtcag ggacgaatcg 540
gagctggtgg agtcctataa tagggcgaaa tcagaggcga aagcggcctt tggcaatgat 600
gaagtttatg tcgaaaagct gattgaaaag ccgaagcaca ttgaagttca agtcatcgga 660
gataaagaag ggaacgtggt tcacctttac gaccgcgact gctctgtgca aaggcgtcat 720
caaaaggtca tcgaagtgaa gccgagcgtt tcgctttctg aatccctccg ggaaaagatt 780
tgcgatgctg ccgttaagct tgcgaagaat gttgaatatg tcaatgccgg tacagtcgaa 840
tttttagttg cgaacgatga gtttttcttt attgaagtga acccgcgtgt tcaggtggag 900
cataccatta cggaaatggt aacgggcgtc gatatcgttc agacgcaaat cctgatcgct 960
gccggtctca gtctggacag cagcgaaatc agcattccga accaggatgc gatcacgctg 1020
cacggatatg cgatccagtc aagggttacg actgaagatc cgtcaaacaa tttcatgcct 1080
gacacaggca aaatcatggc atatcgctca ggcggcggtt tcggggtgag gcttgatacc 1140
ggaaacagtt ttcagggcgc tgtcattacg ccttattacg attcgctgct tgttaagctt 1200
tcaacttggg cgctgacgtt tgaacaggcg gcagccaaaa tggtccgcaa ccttcaggaa 1260
ttcaggatca ggggaatcaa aacgaacatt cctttccttg aaaatgtggc gaaacacgaa 1320
aagtttctta cagggcaata cgacacgtct tttatcgata caacgccaga actttttgtc 1380
tttcctaagc agagagaccg cggaacgaaa atgctgacat atatcgggaa cgtaacggtc 1440
aacggcttcc cgggcatcga taaaaagaag aagcccgagt ttgataagcc gcagatcgtc 1500
aaaacagatg tcgatcagcc aatcgcaagc ggaacaaaac agattcttga tgaacgcgga 1560
gccgaagggc tcgtcaaatg ggtgaaagat caagaggaag tgctcctcac tgatacgacg 1620
ttccgcgacg cccatcagtc attgcttgcg acaagagtca gaacgcatga cctgaaaaaa 1680
atcgccaatc cgacggctgc gctctggcca gagcttttca gtcttgaaat gtggggcggc 1740
gccacatttg atgtcgctta ccgtttcttg aaagaagatc cgtggaaaag gctcgaggag 1800
ctgcggaagg aaattccgaa tacgatgttt caaatgcttt tgagatcttc gaatgccgtc 1860
gggtatacaa actaccctga caacctgatt aaaaagttcg tcagcgaatc ggctgcggcc 1920
ggaatcgatg tattccgcat ttttgacagc ttgaactggg ttaaagggat gacgctcgcc 1980
attgatgcag tgcgcgagtc aggcaagctt gccgaagcgg cgatctgcta tacaggggac 2040
attctcgatc cgaacagaag caagtataat cttgaatatt atacgtcaat ggcaaaagag 2100
cttgaagctg cgggggcgca tattctcggc attaaagata tggccggcct gctgagacct 2160
caagcggcat acgaactggt gtcggctttg aaagagacga tcgacattcc gatccacttg 2220
catacacacg acacgagcgg caacggtatt tttatgtatg cgaaggcgat agaagcaggc 2280
gtcgacatcg tcgacgtagc ggtcagctcg atggcgggtc tgacatcaca gccaagcgca 2340
agctcgcttt accatgcgct tgaaggagat aaacgccgtc cgcagttcaa tgtcgatgcg 2400
gtagagtcgt tgtctcaata ttgggagtct gtcagaaaat attacagcga gtttgagagc 2460
ggcatgattg cgcctcatac cgaaatttac aagcatgaaa tgcctggcgg ccaatacagc 2520
aaccttcagc agcaggctaa gggagtcggc ctcggcgacc gctggaatga agtgaaagaa 2580
atgtacagcc gggtcaacca cctgttcggg gacatcgtaa aggttacgcc gtcatctaaa 2640
gtcgtcgggg atatggcgct ttacatggtg caaaataacc tgacggaaga tgatatttac 2700
gaaaggggag aatctctcga ttttcctgac tcggttgtcg agctttttaa agggtatatc 2760
ggtcagcctc acggcggatt ccctgagaaa ttgcaaaagc tgattttaaa agggcaggag 2820
ccgattacag tacgccctgg agaactgctt gaaccggttt catttgacgc tataaaagcg 2880
gaattcttgg aaaagcatgg tatggagctt tccgaccagg atgctgttgc atatgcgctt 2940
tatccgaaag tgtttaccga gtatgtcaag acagcggaac tgtacgggga tatctctgtg 3000
cttgatacac caaccttcct ctacggcatg accctcggcg aagaaatcga ggtcgagatt 3060
gaaagaggga aaacgctgat cgtcaaactt gtctctatcg gggaaccgcg tccggatgcg 3120
acgagagtcg tttattttga actgaacggc cagcctcgcg aagtggtcat taaagacgaa 3180
agcattaaat cgtccgttca tcaaaaggtg aaagccgacc gttcaaaccc gaatcatatc 3240
gcggcatcta tgccgggaac ggtaatcaag cttcttgtaa gcaaagggga ccaagtgaag 3300
aagggcgatc atttgatgat caatgaagcg atgaaaatgg aaacgaccgt tcaggctccg 3360
ttttcaggaa ctgttgaaaa cattcacgtt acaaacggag aagccattca aaccggcgac 3420
cttctcattg aactgaaaaa ataa 3444
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caacagatag gtgtaaggag gttgagat 28
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tttcacacag gaaacaga 18
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cggtaaagat atcgataagg aggtttttt 29
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aagaaatccg acgccaaagg 20
<210> 8
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atccgctcac aattccacac attatacgag ccggatgatt aattgtcaac ctaacctcgc 60
ctccctactg 70
<210> 9
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caggcggccc tctcgtataa tggcaagtgc ctataatacc cc 42
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcatctagtc ccgcaaactc aa 22
<210> 11
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ctcgtataat gtgtggaatt gtgagcggat aacaacatca gataggtgta aggaggttta 60
gatatgaact cagttgattt gac 83
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cttatcattt ctttccggac gg 22
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccgtccggaa agaaatgata ag 22
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ttatacgaga gggccgcctg 20
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ccagtttaat aagaaaggag acg 23
<210> 16
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttgttatccg ctcacaattc cacacattat acgagccgga tgattaattg tcaatgtcgt 60
gaactgtgag acga 74
<210> 17
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttgaactgaa aaaataaaag tcgaatcagg gctgaagtgg cacactgaat ttg 53
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acaacagacc gagaaagaca ct 22
<210> 19
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aattgtgagc ggataacaac aacagatagg tgtaaggagg ttgagatatg tcacaacagt 60
ctattca 67
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aacgctcggc ttcacttcga t 21
<210> 21
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atcgaagtga agccgagcgt t 21
<210> 22
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ttattttttc agttcaa 17
<210> 23
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
cataagcggg aagggtatcg tg 22
<210> 24
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ttgttatccg ctcacaattc cacacattat acgagccgga tgattaattg tcaattgaac 60
cgtgccgcca ttctc 75
<210> 25
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgatgatgaa tcatcagtaa accgtataag ccgcatgtcg agatggcatg c 51
<210> 26
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atgtcgtgag cgtggtattg tc 22
<210> 27
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gaattgtgag cggataacaa cggtaaagat atcgataagg aggtttttta tgcgagtgtt 60
gaagttcgg 69
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aaacacgggt ccacgttgtc 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gacaacgtgg acccgtgttt 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttactgatga ttcatcatca 20
<210> 31
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gttttagagc tagaaatagc aagttaa 27
<210> 32
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
attataccta ggactgagc 19
<210> 33
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
agtcctaggt ataatactag taacacagca atacggtacg cgttttagag ctagaa 56
<210> 34
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ttctagctct aaaacgcgta ccgtattgct gtgttactag tattatacct aggact 56
<210> 35
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
agtcctaggt ataatactag ttatctgacc agtaaatggg agttttagag ctagaa 56
<210> 36
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ttctagctct aaaactccca tttactggtc agataactag tattatacct aggact 56
<210> 37
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
agtcctaggt ataatactag tgcctatccg ggctgtcccg agttttagag ctagaa 56
<210> 38
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ttctagctct aaaactcggg acagcccgga taggcactag tattatacct aggact 56
Claims (10)
1.一种大肠杆菌重组核酸,其特征在于,所述重组核酸包括磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因。
2.根据权利要求1所述重组核酸,其特征在于,所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因、丙酮酸羧化酶pyc的编码基因和苏氨酸操纵子的编码基因均由Trc启动子启动表达。
3.根据权利要求1所述重组核酸,其特征在于,所述磷酸烯醇式丙酮酸羧化激酶pck的编码基因来源于枯草芽孢杆菌;
所述丙酮酸羧化酶pyc的编码基因来源于地衣芽孢杆菌。
4.根据权利要求1或3所述重组核酸,其特征在于,所述磷酸烯醇式丙酮酸羧化激酶pck经RBS优化和143位甘氨酸突变为精氨酸;
所述丙酮酸羧化酶pyc经RBS优化和247位丙氨酸突变为赖氨酸。
5.根据权利要求1所述重组核酸,其特征在于,所述苏氨酸操纵子经RBS优化和144位丙氨酸突变为天冬氨酸。
6.一种包含权利要求1~5任一项所述重组核酸的重组大肠杆菌,所述重组大肠杆菌过表达磷酸烯醇式丙酮酸羧化激酶pck、丙酮酸羧化酶pyc和苏氨酸操纵子。
7.根据权利要求6所述重组大肠杆菌,其特征在于,所述重组大肠杆菌的基础菌株包括大肠杆菌K-12 W3110。
8.权利要求6或7所述重组大肠杆菌的培养方法,其特征在于,包括以下步骤:将所述重组大肠杆菌接种于种子培养基上进行培养,得种子液;所述种子培养基包括以下浓度的组分:玉米浆干粉5g/L,葡萄糖20g/L,酵母粉5g/L,KH2PO4 2g/L,硫酸镁1g/L,FeSO4·7H2O20mg/L和MnSO4·H2O 20mg/L。
9.权利要求6或7所述重组大肠杆菌在生物合成L-苏氨酸中的应用,所述重组大肠杆菌以葡萄糖为发酵底物。
10.一种生物合成L-苏氨酸的方法,其特征在于,包括以下步骤:将利用权利要求8所述培养方法得到的种子液接种于发酵培养基中,进行有氧发酵,发酵液中含L-苏氨酸;
所述发酵培养基包括以下浓度的组分:葡萄糖20g/L,磷酸二氢钾2g/L,酵母粉3g/L,甜菜碱1g/L,硫酸镁1g/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,玉米浆干粉8g/L和维生素B1 10mg/L。
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US17/930,223 US20240018557A1 (en) | 2022-05-19 | 2022-09-07 | Recombinant nucleic acid of escherichia coli, recombinant escherichia coli and culturing method thereof, and method for biosynthesizing l-threonine thereby |
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CN118406630A (zh) * | 2024-06-26 | 2024-07-30 | 哈尔滨象柏生物科技有限公司 | 一种生产l-苏氨酸的基因工程菌及其制备方法和发酵工艺 |
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US20030087381A1 (en) * | 1998-04-13 | 2003-05-08 | University Of Georgia Research Foundation, Inc. | Metabolically engineered organisms for enhanced production of oxaloacetate-derived biochemicals |
CN111019878A (zh) * | 2020-01-13 | 2020-04-17 | 江南大学 | L-苏氨酸产量提高的重组大肠杆菌及其构建方法与应用 |
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US20030087381A1 (en) * | 1998-04-13 | 2003-05-08 | University Of Georgia Research Foundation, Inc. | Metabolically engineered organisms for enhanced production of oxaloacetate-derived biochemicals |
CN111019878A (zh) * | 2020-01-13 | 2020-04-17 | 江南大学 | L-苏氨酸产量提高的重组大肠杆菌及其构建方法与应用 |
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CN118406630A (zh) * | 2024-06-26 | 2024-07-30 | 哈尔滨象柏生物科技有限公司 | 一种生产l-苏氨酸的基因工程菌及其制备方法和发酵工艺 |
CN118406630B (zh) * | 2024-06-26 | 2024-09-24 | 哈尔滨象柏生物科技有限公司 | 一种生产l-苏氨酸的基因工程菌及其制备方法和发酵工艺 |
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