CN117165565A - 苏氨酸脱水酶突变体、产支链氨基酸的重组微生物及其应用 - Google Patents
苏氨酸脱水酶突变体、产支链氨基酸的重组微生物及其应用 Download PDFInfo
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- CN117165565A CN117165565A CN202110998231.6A CN202110998231A CN117165565A CN 117165565 A CN117165565 A CN 117165565A CN 202110998231 A CN202110998231 A CN 202110998231A CN 117165565 A CN117165565 A CN 117165565A
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- threonine dehydratase
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Abstract
本发明涉及微生物技术领域,具体涉及苏氨酸脱水酶突变体、产支链氨基酸的重组微生物及其应用。本发明提供苏氨酸脱水酶突变体,以谷氨酸棒杆菌野生型苏氨酸脱水酶为参考序列,其含有第54位、第131位、第171位、第218位、第243位、第381、第383位突变中的任意一种或至少两种的组合。该突变体能够有效解除异亮氨酸对苏氨酸脱水酶的反馈抑制,显著提高在高浓度异亮氨酸存在时苏氨酸脱水酶的催化活性,提高微生物的异亮氨酸等支链氨基酸的合成能力、产量和转化率。本发明提供的表达该突变体的重组微生物能够高效地合成异亮氨酸,异亮氨酸的产量和转化率均显著提高。
Description
技术领域
本发明涉及微生物技术领域,具体涉及苏氨酸脱水酶突变体、产支链氨基酸的重组微生物及其应用。
背景技术
支链氨基酸(branch chain amino acid,BCAA)包括缬氨酸、亮氨酸和异亮氨酸,是人体的必需氨基酸,在人体内具有重要的生理功能,广泛应用于食品、药品、保健品、化妆品、饲料等领域,可作为人体营养增强剂、动物饲料添加剂、医疗产品成分和化妆品成分等。目前,支链氨基酸主要通过微生物发酵生产,大肠杆菌和谷氨酸棒杆菌是主要的生产菌株。但是,大肠杆菌通常会合成内毒素,可能会造成产品污染。因此,谷氨酸棒状杆菌成为生产支链氨基酸的理想菌株。
以异亮氨酸的合成途径为例,在谷氨酸棒状杆菌中,从L-天冬氨酸开始,经过十步反应合成异亮氨酸,其中部分合成酶也可用于合成缬氨酸和亮氨酸。因此,高产异亮氨酸的菌株不易获得。
为了改善菌株的L-异亮氨酸生产能力,多采取诱变育种的方法,但是该方法得到的菌株传代后易退化。例如:专利申请CN111197014A公开了通过紫外照射和亚甲基双胍双诱变的方式获得一株L-异亮氨酸生产菌(保藏编号为CCTCC NO:M 2019695),但该生产菌在培养过程中需要添加新霉素防止回复突变。现有技术中也有利用基因工程理性改造谷氨酸棒状杆菌用于产L-异亮氨酸的报道(例如:专利申请CN111321100A公开了一株L-异亮氨酸生产菌,产酸32.1g/L,转化率18.1%,发酵周期70-75h),但是转化率仍较低且发酵周期较长,无法满足工业生产的需求。
发明内容
本发明的目的之一是提供苏氨酸脱水酶突变体及其应用。本发明的另一目的是提供表达苏氨酸脱水酶突变体的重组微生物及其应用。
具体地,本发明提供以下技术方案:
本发明提供苏氨酸脱水酶突变体,以谷氨酸棒杆菌野生型苏氨酸脱水酶为参考序列,所述苏氨酸脱水酶突变体含有第54位由丙氨酸突变为除丙氨酸以外的氨基酸、第131位由缬氨酸突变为除缬氨酸以外的氨基酸、第171位由精氨酸突变为除精氨酸以外的氨基酸、第218位由蛋氨酸突变为除蛋氨酸以外的氨基酸、第243位由甘氨酸突变为除甘氨酸以外的氨基酸、第381位由苏氨酸突变为除苏氨酸以外的氨基酸、第383位由苯丙氨酸突变为除苯丙氨酸以外的氨基酸的突变中的任意一种或至少两种的组合。
本发明中,苏氨酸脱水酶催化苏氨酸脱水分解生成氨和α-酮丁酸的反应,该酶受到异亮氨酸的反馈抑制调控。谷氨酸棒杆菌中苏氨酸脱水酶由ilvA基因编码,野生型谷氨酸棒杆菌的苏氨酸脱水酶的氨基酸序列如SEQ ID NO.1所示。
本发明发现,苏氨酸脱水酶的第54位、第131位、第171位、第218位、第243位、第381位、第383位氨基酸的突变能够解除异亮氨酸对该酶的反馈抑制,显著提高在异亮氨酸存在条件下该酶的活性,进而提高异亮氨酸等支链氨基酸的合成能力。
优选地,以谷氨酸棒杆菌野生型苏氨酸脱水酶为参考序列,所述苏氨酸脱水酶突变体含有第54位由丙氨酸突变为苏氨酸、第131位由缬氨酸突变为丙氨酸、第171位由精氨酸突变为组氨酸、第218位由蛋氨酸突变为异亮氨酸、第243位由甘氨酸突变为精氨酸、第381位由苏氨酸突变为丙氨酸、第383位由苯丙氨酸突变为丙氨酸的突变中的任意一种或至少两种的组合。
进一步优选地,所述苏氨酸脱水酶突变体具体如SEQ ID NO.2所示的氨基酸序列。该突变体对于异亮氨酸反馈抑制接触的效果更优,能够显著提高在高浓度异亮氨酸存在条件下该酶的活性,进而促进异亮氨酸等支链氨基酸的高效合成。
本领域技术人员应该理解,在上述苏氨酸脱水酶突变体序列的N端或C端添加标签蛋白或将其与其他蛋白进行融合形成融合蛋白,在不改变上述苏氨酸脱水酶突变体本身结构的情况下,其活性不会发生明显改变,因此,上述添加标签的蛋白或融合蛋白也在本发明的保护范围内。
本发明还提供编码所述苏氨酸脱水酶突变体的基因。
根据上述提供的苏氨酸脱水酶突变体的氨基酸序列,本领域技术人员能够获得其编码基因的序列。基于密码子的简并性,编码上述氨基酸序列的基因序列不止一种,所有能够编码上述突变体的基因均在本发明的保护范围内。
作为本发明的一种实施方式,编码苏氨酸脱水酶突变体的基因的核苷酸序列如SEQ ID NO.3所示。
本发明还提供含有所述基因的生物材料,所述生物材料为重组DNA、载体或宿主细胞。
其中,所述重组DNA可为在所述基因的上游或下游连接用于驱动其转录、翻译的元件得到的重组DNA。所述载体可为表达载体或克隆载体,包括但不限于质粒载体、噬菌体载体、转座子等。所述宿主细胞可为微生物细胞。
进一步地,本发明提供一种重组微生物,其表达所述苏氨酸脱水酶突变体。
以上所述的表达苏氨酸脱水酶突变体可通过在染色体上整合苏氨酸脱水酶突变体的编码基因的方式实现,也可通过转入携带苏氨酸脱水酶突变体的编码基因的表达载体的方式实现。
对于染色体整合的位置和表达载体的种类,本发明不做特殊限定,只要能够实现苏氨酸脱水酶突变体的表达即可。
对于出发菌株中自身含有的苏氨酸脱水酶编码基因,可以将其失活,或者将其直接替换为编码苏氨酸脱水酶突变体的基因。
作为本发明的一种优选实施方式,将染色体上原始的苏氨酸脱水酶编码基因替换为编码所述苏氨酸脱水酶突变体的基因。
为进一步增强苏氨酸脱水酶突变体的表达水平,优选在所述苏氨酸脱水酶突变体的编码基因的上游可操作性地连接优化的ilvA启动子,所述优化的ilvA启动子为将原始ilvA启动子的-10区(TACACT)突变为TATAAT得到。
优选地,所述优化的ilvA启动子的核苷酸序列如SEQ ID NO.4所示;所述重组微生物中,在原始ilvA启动子与ilvA编码基因之间插入优化的ilvA启动子。
与出发菌株相比,以上重组微生物的L-异亮氨酸的末端合成路径被显著强化,异亮氨酸等支链氨基酸的产量和转化率均显著提高。
为进一步提高异亮氨酸等支链氨基酸的合成能力,可对上述重组微生物进一步改造。
优选地,与出发菌株相比,所述重组微生物中,磷酸烯醇式丙酮酸羧化酶的表达和/或酶活性增强。
磷酸烯醇式丙酮酸羧化酶(ppc)催化磷酸烯醇式丙酮酸生成支链氨基酸的合成前体草酰乙酸。本发明发现,在苏氨酸脱水酶改造的重组微生物中,以其他改造靶点相比,增强磷酸烯醇式丙酮酸羧化酶的表达、酶活性能够显著提高异亮氨酸合成途径的代谢通量,促进异亮氨酸等支链氨基酸的高效合成。
进一步地,与出发菌株相比,所述重组微生物中,6-磷酸葡萄糖脱氢酶的表达和/或酶活性增强。
6-磷酸葡萄糖脱氢酶(gnd)催化6-磷酸葡萄糖生成6-磷酸葡萄糖酸-δ-内酯,在此过程中产生还原力。本发明发现,在苏氨酸脱水酶改造的重组微生物中,或者在苏氨酸脱水酶和磷酸烯醇式丙酮酸羧化酶联合改造的重组微生物中,增强6-磷酸葡萄糖脱氢酶的表达、酶活性,增加还原力NADPH的供应,能够更显著地提升异亮氨酸等支链氨基酸的合成能力。
进一步地,与出发菌株相比,所述重组微生物中,乙酰羟基酸合酶和二羟酸还原异构酶的表达和/或酶活性增强。
乙酰羟基酸合酶(ilvBN)和二羟酸还原异构酶(ilvC)是异亮氨酸合成途径的催化酶,在上述靶点改造的重组微生物中,进一步增强异亮氨酸末端合成途径的催化酶的表达、酶活性更有利于异亮氨酸的合成和积累。
在表达苏氨酸脱水酶突变体的重组微生物中,以上改造靶点可以进行不同的组合,例如:在表达苏氨酸脱水酶突变体的重组微生物中,增强磷酸烯醇式丙酮酸羧化酶的表达和/或酶活性增强,或者,在表达苏氨酸脱水酶突变体的重组微生物中,增强6-磷酸葡萄糖脱氢酶的表达和/或酶活性,或者同时增强磷酸烯醇式丙酮酸羧化酶、6-磷酸葡萄糖脱氢酶的表达和/或酶活性,或者,在表达苏氨酸脱水酶突变体的重组微生物中,增强乙酰羟基酸合酶和二羟酸还原异构酶的表达和/或酶活性或者同时增强磷酸烯醇式丙酮酸羧化酶、6-磷酸葡萄糖脱氢酶、乙酰羟基酸合酶和二羟酸还原异构酶的表达和/或酶活性。
以上所述的表达的增强通过以下任一种方式实现:
(1)增加出发菌株中目标酶编码基因的拷贝数;
(2)将出发菌株中目标酶编码基因的表达调控元件替换为活性更强的调控元件;
(3)优化出发菌株中目标酶编码基因的密码子。
上述(1)中,增加拷贝数可通过在染色体上整合额外的基因拷贝,或者导入携带目标基因的表达质粒实现。
上述(2)中,表达调控元件包括但不限于启动子、核糖体结合位点(RBS)、增强子等。其中活性更强的启动子包括但不限于Ptac、Plac、Ptrp、Ptrc。
酶活性的增强通过以下任一种方式实现:
(1)将出发菌株中目标酶编码基因的一个或多个碱基进行突变,以使得目标酶的酶活性增强;
(2)将出发菌株中目标酶的编码基因替换为其他生物来源的目标酶编码基因,以使得目标酶的酶活性增强。
作为本发明的一种优选实施方式,本发明提供以下重组微生物:
将出发菌株中的苏氨酸脱水酶编码基因替换为编码所述苏氨酸脱水酶突变体的基因,并以SEQ ID NO.4所示的启动子起始编码所述苏氨酸脱水酶突变体的基因的转录;并且,与出发菌株相比,该重组微生物的染色体上额外整合一个ppc基因的拷贝,以Ptac为该ppc基因的启动子;并且该重组微生物的gnd基因的启动子由原始gnd基因启动子替换为Ptac启动子,并且,该重组微生物携带表达ilvBNC基因的表达质粒。
优选地,所述重组微生物为棒杆菌属或短杆菌属细菌。
其中,所述棒杆菌属细菌优选为选自谷氨酸棒杆菌(Corynebacteriumglutamicum)、北京棒杆菌(Corynebacterium pekinense)、有效棒杆菌(Corynebacteriumefficiens)、钝齿棒杆菌(Corynebacterium crenatum)、嗜热产氨棒杆菌(Corynebacterium thermoaminogenes)、产氨棒杆菌(Corynebacterium aminogenes)的细菌。所述短杆菌属细菌优选为选自黄色短杆菌(Breviabacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)的细菌。
本发明中,所述出发菌株优选为能够合成、积累异亮氨酸等支链氨基酸的细菌。
作为本发明的一种实施方式,所述出发菌株为MHZ-1012-2。MHZ-1012-2菌株已于2016年11月30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(CGMCC),保藏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,保藏编号为CGMCCNo.13406,分类命名为:谷氨酸棒杆菌Corynebacterium glutamicum,该菌株公开于专利申请CN201611250330.1中。
本发明提供的上述重组微生物可采用本领域常规的方法构建得到,例如:其中的编码基因的突变、替换、启动子的替换、染色体整合额外基因拷贝可通过同源重组的方法进行。
基于上述苏氨酸脱水酶突变体、重组微生物的功能,本发明提供所述苏氨酸脱水酶突变体或所述基因或所述生物材料或所述重组微生物的如下任一种应用:
(1)在工程菌构建中的应用;
(2)在发酵生产支链氨基酸或其衍生物中的应用;
(3)在提高菌株的支链氨基酸或其衍生物的产量和/或转化率中的应用。
优选地,以上(1)中所述的应用中,所述工程菌为支链氨基酸(尤其是异亮氨酸)的工程菌。优选为谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)、黄色短杆菌(Breviabacterium flavum)。
本发明的支链氨基酸的衍生物为以异亮氨酸、缬氨酸、亮氨酸为前体合成的物质。在重组微生物合成支链氨基酸的能力显著提高的情况下,以支链氨基酸为前体的下游产物的合成能力也会增强。其中,异亮氨酸的衍生物包括但不限于4-羟基异亮氨酸、N-乙酰-DL-缬氨酸、2-氨基丁酸、2-羟基酮丁酸等,缬氨酸的衍生物包括但不限于泛解酸、2-氨基异戊酸等,亮氨酸的衍生物包括但不限于N-乙酰-L-亮氨酸等。
本发明提供一种发酵生产支链氨基酸或其衍生物的方法,包括:培养所述重组微生物得到培养物,将培养物经分离提取得到支链氨基酸或其衍生物。
具体地,所述发酵生产支链氨基酸或其衍生物的方法包括:将所述重组微生物接种于种子培养基中进行种子培养,得到种子液,将种子液接种于发酵培养基中培养,得到发酵液,将发酵液经分离提取得到所述氨基酸或其衍生物。
优选地,所述发酵培养基包括如下组分:葡萄糖80-120g/L、(NH4)2SO4 5-20g/L、磷酸氢二钾0.5-2g/L、硫酸镁0.3-0.8g/L、玉米浆20-40g/L,生物素0.05-0.2mg/L、VB1 0.05-0.2mg/L,pH 6.8-7.2。
优选地,所述种子培养基包括如下组分:葡萄糖10-30g/L、氯化钾0.3-0.8g/L、硫酸镁0.3-0.8g/L、玉米浆50-70g/L、生物素0.2-0.5mg/L、VB1 1-3mg/L,pH 6.8-7.2。
优选地,所述发酵的温度为30-33℃。
本发明中,所述支链氨基酸优选为异亮氨酸。
本发明的有益效果在于:本发明提供的苏氨酸脱水酶突变体能够有效解除异亮氨酸对苏氨酸脱水酶的反馈抑制,显著提高在高浓度异亮氨酸存在时,苏氨酸脱水酶的催化活性,提高微生物的异亮氨酸等支链氨基酸的合成能力、产量和转化率。
本发明提供的表达苏氨酸脱水酶突变体的重组微生物能够高效地合成异亮氨酸等支链氨基酸,异亮氨酸的产量和转化率均显著提高。
本发明通过多种代谢工程策略结合构建的异亮氨酸工程菌MHI-4/pEKEX2-ilvBNMC,在发酵罐补料分批发酵中可积累36.2g/L的L-异亮氨酸,糖酸转化率达到0.276g/g,同时,副产物L-亮氨酸、L-缬氨酸和L-苏氨酸的含量显著减少。该菌株是目前报道中非常有竞争力的工业生产菌,为L-异亮氨酸的工业化生产奠定基础。
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。
以下实施例中所涉及的试剂和材料均为市售产品,均可以通过商业渠道购买获得。其中,出发菌株MHZ-1012-2为谷氨酸棒杆菌(Corynebacterium glutamicum),于2016年11月30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(CGMCC),保藏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,保藏编号为CGMCCNo.13406。普通液体脑心浸液培养基配方为3.7%脑心浸粉溶液,普通固体脑心浸液培养基配方为3.7%脑心浸粉溶液和1.8%琼脂粉。
以下实施例中使用的引物序列如表1所示。
表1 引物序列
实施例1 ilvA基因突变和强化ilvA基因表达
以MHZ-1012-2为出发菌株,将MHZ-1012-2中ilvA基因突变为编码SEQ ID NO.2所示的苏氨酸脱水酶突变体的基因,并在原始ilvA启动子与ilvA编码基因之间插入优化的启动子,以强化ilvA基因表达,具体方法如下:
采用全基因合成的方式合成包含ilvA优化后的启动子(SEQ ID NO.4)和ilvA突变后的编码区(ORF)序列(SEQ ID NO.3)的DNA片段(SEQ ID NO.5)。将出发菌株MHZ-1012-2中原有的ilvA基因编码区序列替换为上述合成的DNA片段,即在原始ilvA启动子与ilvA编码基因之间插入优化的启动子,同时将ilvA编码区替换为突变后的编码区,具体方法如下:
以含有SEQ ID NO.5所述序列的载体为模板,以PI-ilvA-2f/PI-ilvA-2r为引物扩增得到ilvA优化后的启动子部分和突变后的ORF区序列,命名为片段2(1591bp)。分别以引物对PI-ilvA-1f/PI-ilvA-1r和PI-ilvA-3f/PI-ilvA-3r以菌株MHZ-1012-2基因组为模板扩增得到上同源臂和下同源臂,分别命名为片段1(855bp)和片段3(844bp)。利用引物对PI-ilvA-1f/PI-ilvA-3r,通过融合PCR将3个片段进行融合,并利用内切酶XbaI和BamHI将融合片段和载体pK18-mob-sacB进行双酶切并进行回收。利用T4连接酶进行连接并转化入T1感受态。将获得测序正确的重组质粒电转化菌株MHZ-1012-2感受态,进过两轮筛选得到出发菌株MHZ-1012-2中原有的ilvA基因启动子及其编码区序列替换为SEQ ID NO.5所示序列的改造菌,取代的适当性主要通过在对应于修饰序列(PI-ilvA-ID-F/PI-ilvA-ID-R)的引物组合中选择扩增菌株来确定。另外,通过使用引物PI-ilvA-F和PI-ilvA-R分析修饰的序列,以二次确认取代的适当性,将测序正确的菌株命名为MHI-1。
实施例2 含苏氨酸脱水酶突变体的菌株构建
以MHZ-1012-2为出发菌株,将MHZ-1012-2中ilvA基因突变为编码SEQ ID NO.2所示的苏氨酸脱水酶突变体的基因,具体方法如下:
以实施例1中全基因合成的片段为模板,利用引物对PI-ilvAM-2f/PI-ilvAM-2r扩增获得含突变位点Ala54Thr,Val131Ala,Arg171His,Met218Ile,Gly243Arg,Thr381Ala,Phe383Ala的ilvA基因。以菌株MHZ-1012-2的基因组为模板,利用引物对PI-ilvATM-1f/PI-ilvATM-1r扩增获得上同源臂;以菌株MHZ-1012-2的基因组为模板,利用引物对PI-ilvATM-3f/PI-ilvATM-3r扩增获得下同源臂;以3个片段的混合物为模板,利用引物对PI-ilvATM-1f/PI-ilvATM-3r进行融合PCR并获得含ilvA突变体的融合片段。融合片段与pK18-mob-sacB载体用XbaI和BamHI进行双酶切。将两个酶切产物以T4 DNA Ligase连接1h,连接产物转化Trans1 T1感受态细胞,得到重组质粒pK18mobsacB-ilvAM。将测序正确的重组质粒电转入菌株MHZ-1012-2的感受态细胞中,进过两轮筛选得到含ilvA突变体的改造菌,通过使用引物PL-ilvATM-F和PL-ilvATM-R分析修饰的序列,以二次确认取代的适当性,并命名为MHI-2。
实施例3 增加ppc基因的拷贝数
在实施例1构建的MHI-1菌株中进一步在位点cg1507处整合以Ptac为启动子的ppc基因,以强化ppc基因的表达,具体方法如下:
以菌株MHZ-1012-2基因组为模板、以引物对PI-ppc-1f/PI-ppc-1r扩增上同源臂,命名为片段1(1024bp);以基因组为模板、以引物对PI-ppc-2f/PI-ppc-2r扩增ppc基因及其终止子序列,命名为片段2(2907bp);以pXMJ19为模板、以引物对PI-ppc-3f/PI-ppc-3r扩增启动子Ptac,命名为片段3(307bp);以菌株MHZ-1012-2基因组为模板、以引物对PI-ppc-4f/I-ppc-4r扩增下同源臂,命名为片段4(930bp);利用引物对PI-ppc-1f/PI-ppc-4r以片段1-4为模板得到重叠片段;利用引物对PI-pK18-F/PI-pK18-R将载体pK18-mob-sacB进行线性化,然后在组装试剂盒的作用下完成重叠片段与线性载体的连接,转化T1感受态。将测序正确的重组质粒电转化至菌株MHI-1感受态,经过两轮筛选得到ppc基因整合的菌株;整合的适当性主要通过在对应于修饰序列(PI-ppc-ID-F/PI-ppc-4r)的引物组合中选择扩增菌株来确定。另外,通过使用引物PI-ppc-F和PI-ppc-R进行所选菌株的序列的分析,以二次确认取代的适当性,将测序正确的菌株命名为MHI-3。
实施例4 强化gnd基因的表达
在实施例3构建的MHI-3菌株中进一步将gnd基因的原有启动子替换为强启动子Ptac,以强化gnd基因的表达,具体方法如下:
以菌株MHZ-1012-2基因组为模板、以引物对PI-gnd-1f/PI-gnd-1r扩增上同源臂,大小986bp;以pXMJ19为模板、以引物对PI-gnd-2f/PI-gnd-2r扩增Ptac,大小289bp;以基因组为模板、以引物对PI-gnd-3f/PI-gnd-3r扩增下同源臂,大小884bp;利用重叠PCR将3个片段进行融合,并利用酶切位点BamHI/EcoRI将融合片段与pK18-mob-sacB载体连接,转化T1感受态。将测序正确的重组质粒电转入菌株MHI-3的感受态中,经两轮筛选得到gnd基因改造菌,整合的适当性主要通过在对应于修饰序列(PI-gnd-ID-F/PI-ppc-3r)的引物组合中选择扩增菌株来确定。另外,通过使用引物PI-gnd-F和PI-gnd-R进行所选菌株的序列的分析,以二次确认取代的适当性,将测序正确的菌株命名为MHI-4。
实施例5 质粒过表达ilvBNC
在实施例4构建的菌株MHI-4中,进一步导入过表达ilvBNC的质粒,具体方法如下:
以MHZ-1012-2基因组为模板、利用引物对PI-ilvBNC-F/PI-ilvBNC-R扩增得到PilvB-ilvBNC片段。利用酶切位点BamHI/EcoRI将PilvB-ilvBNC片段和质粒pEKEX2连接,并转化T1感受态,最终获得测序正确的表达质粒pEKEX2-ilvBNC。
将质粒pEKEX2-ilvBNC电转入菌株MHI-4的感受态,获得最终菌株MHI-4/pEKEX2-ilvBNC。
实施例6 苏氨酸脱水酶突变体的活性测定
使用引物PI-ilvA-f/PI-ilvA-r分别以MHZ-1012-2和实施例1的全基因合成的片段为模板扩增得到野生型ilvA基因和突变型ilvA基因,利用EcoRI和HindIII将ilvA基因片段与载体pET28a连接。将构建好的载体pET28a-ilvATM和pET28a-ilvA转入E.coli BL21(DE3)。利用0.5mM的IPTG进行诱导表达,并利用文献(Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum)报道的方法对MHZ-1012-2的苏氨酸脱水酶和本发明的苏氨酸脱水酶突变体(氨基酸序列如SEQ ID NO.2所示)进行纯化和酶活测定。
酶活检测结果如表2所示,其中,相对酶活性为以野生型苏氨酸脱水酶未添加异亮氨酸时的活性计为100%计算得到。结果显示,序列如SEQ ID NO.2所示的苏氨酸脱水酶突变体基本解除了异亮氨酸的反馈抑制,在高浓度异亮氨酸存在时,仍能够保留较高的酶活性,而且,在异亮氨酸不存在时,SEQ ID NO.2所示的苏氨酸脱水酶突变体的酶活性较野生型提高了28.9%。
表2 酶活性检测结果
实施例7 摇瓶发酵验证
将实施例1-5构建得到的菌株MHI-1、MHI-2、MHI-3、MHI-4、MHI-4/pEKEX2-ilvBNC以及出发菌株MHZ-1012-2进行摇瓶发酵并比较L-异亮氨酸的生产性能。
摇瓶发酵使用的培养基如下:
固体活化平板:BHI培养基37g/L;琼脂粉20g/L;
种子培养基:葡萄糖20g/L,蛋白胨10g/L,酵母膏5g/L,尿素1.5g/L,KH2PO4 4g/L,K2HPO4 8g/L,MgSO4·7H2O 0.5g/L,生物素100μg/L,盐酸硫胺素1000μg/L,泛酸钙2000μg/L,烟酰胺2000μg/L,pH调至7.0。
发酵培养基:葡萄糖100g/L、豆粕提取物9.75g/L、玉米浆干粉14.4g/L、MgSO4·7H2O 2g/L、KH2PO4·12H2O 2g/L、FeSO4·7H2O 0.01g/L、MnSO4·H2O 0.01g/L、VB1 0.01g/L、(NH4)2SO4 50g/L,蒸馏水配制,pH调至7.0。
其中,菌株MHI-4/pEKEX2-ilvBNC的培养需添加25mg/L的卡那霉素。
摇瓶发酵的方法如下:
种子培养:在平板上刮取1环菌株MHI-1、MHI-2、MHI-3、MHI-4、MHI-4/pEKEX2-ilvBNC以及出发菌株MHZ-1012-2分别接种到含有50ml种子培养基的锥形瓶(500ml)中,然后在30-31℃条件下、以110rpm振荡培养15-17h至OD值为16-18;
摇瓶发酵:按10%的接种量转接至装有25ml发酵培养基的锥形瓶(500ml)中,然后在30-31℃条件下、以135rpm振荡培养持续48小时。培养后使用HPLC检测发酵液中L-异亮氨酸以及其他氨基酸的含量。发酵结果见如表3所示。结果显示,与出发菌株相比,菌株MHI-1的L-异亮氨酸产量提高了1.8倍,同时副产物L-亮氨酸和L-缬氨酸的含量明显降低,表明针对ilvA基因的改造方式有效,将代谢流由缬氨酸和亮氨酸途径拉向异亮氨酸合成途径。菌株MHI-2的L-异亮氨酸的产量较出发菌提高了54.2%,表明ilvA突变体对于L-异亮氨酸的产量提高具有明显的促进作用,并且ilvA突变体的酶活性测定结果佐证了该作用。MHI-3、MHI-4、MHI-4/pEKEX2-ilvBNC菌株的L-异亮氨酸的产量在菌株MHI-1的基础上进一步提高,分别提高了56.7%、92.5%倍和2倍,表明增加前体供应和还原力供应对于提高L-异亮氨酸的产量具有显著的促进作用。
表3 摇瓶发酵结果
实施例8 发酵罐发酵验证
将出发菌株MHZ-1012-2和工程菌MHI-4/pEKEX2-ilvBNC在50-L BAILUN发酵罐上进行补料分批发酵,具体方法如下:
在2个固体活化平板上同时活化出发菌株MHZ-1012-2和工程菌MHI-4/pEKEX2-ilvBNC,30℃静置培养36-48h;菌苔转接入2个装有50ml种子培养基的500-ml摇瓶中,于30℃、200rpm培养6-8h;按体积分数0.5%的接种量,转接100ml种子培养物至装有20L种子培养基的50-L种子罐中,在搅拌200-800rpm、温度31-33℃条件下培养;然后按接种比1%将2L种子转接至装有20L发酵培养基的50-L发酵罐中。
其中,种子培养基配方如下:葡萄糖20g/L、玉米浆60g/L、氯化钾0.5g/L、硫酸镁0.5g/L、消泡剂0.3mL/L、生物素0.3mg/L、VB1 2mg/L,其余为水。
发酵培养基配方如下:葡萄糖80g/L、玉米浆30g/L、硫酸铵10g/L、磷酸氢二钾1g/L、硫酸镁0.5g/L、消泡剂0.3mL/L、生物素0.1mg/L、VB1 0.1mg/L,其余为水。
发酵条件为:罐压0.05-0.1Mpa,通气量10-25L/min,搅拌200-800rpm,温度31-33℃,pH 6.8-7.2。控制发酵液中残糖浓度在0.5g/L以下,耗糖速率维持在6g/L/h。当杂酸(包括缬氨酸、亮氨酸和苏氨酸等)含量达到1g/L时,以0.3g/L/h的速度逐步降低耗糖速率直至发酵48h放罐。使用HPLC检测发酵液中L-异亮氨酸以及其他氨基酸的含量,发酵结果见表4。
表4 发酵罐发酵结果
以上结果表明,本发明所构建的基因工程菌MHI-4/pEKEX2-ilvBNC能够实现发酵过程中L-异亮氨酸的高效合成和积累,同时,副产物L-亮氨酸、L-缬氨酸和L-苏氨酸的含量显著降低,该菌株具有较好的工业应用前景。
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。
序列表
<110> 胡丹
<120> 苏氨酸脱水酶突变体、产支链氨基酸的重组微生物及其应用
<130> KHP211120023.2
<160> 44
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atgagtgaaa catacgtgtc tgagaaaagt ccaggagtga tggctagcgg agcggagctg 60
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ccattgcagt attgccctcg tctttctgag gaaaccggaa cggaaatcta ccttaagcgt 180
gaggatctgc aggatgttcg ttcctacaag atccgcggtg cgctgaactc tggagcgcag 240
ctcactcagg agcagcgcga tgcaggtatc gttgccgcat ctgcaggtaa ccatgcccag 300
ggcgtggcct atgtgtgcaa gtccttgggc gttcagggac gcatctatgt tcctgtgcag 360
actccaaagc aaaagcgtga ccgcatcatg gctcacggcg gagagtttgt ctccttggtg 420
gtcactggca ataacttcga cgaagcatcg gctgcagcgc atgaagatgc agagcgcacc 480
ggcgcaacgc tgatcgagcc tttcgatgct cacaacaccg tcatcggtca gggtacagtg 540
gctgctgaga tcttgtcgca gctgacttcc atgggcaaga gtgcagatca cgtgatggtt 600
ccagtcggcg gtggcggact tcttgcaggt gtggtcagct acatggctga tatagcacct 660
cgcactgcga tcgttggtat cgaaccagcg ggagcagcat ccatgcaggc tgcattgcac 720
aatggtagac caatcacttt ggagactgtt gatccctttg tggacggcgc agcagtcaaa 780
cgtgtcggag atctcaacta caccatcgtg gagaagaacc agggtcgcgt gcacatgatg 840
agcgcgaccg agggcgctgt gtgtactgag atgctcgatc tttaccaaaa cgaaggcatc 900
atcgcggagc ctgctggcgc gctgtctatc gctgggttga aggaaatgtc ctttgcacct 960
ggttctgtcg tggtgtgcat catctctggt ggcaacaacg atgtgctgcg ttatgcggaa 1020
atcgctgagc gctccttggt gcaccgcggt ttgaagcact acttcttggt gaacttcccg 1080
caaaagcctg gtcagttgcg tcacttcctg gaagatatcc tgggaccgga tgatgacatc 1140
gcgctggcag agtacctcaa gcgcaacaac cgtgagaccg gtactgcgtt ggtgggtatt 1200
cacttgagtg aagcatcagg attggattct ttgctggaac gtatggagga atcggcaatt 1260
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gcgtcttaat ggtgcgcgtg atgatgggtc tgtacaggag agggttggtg gtgtagcgtc 180
gctgttaagt gcttgagtta gcacgtgttc ttatcgcccc cctggttcac aaacccctgg 240
cagcgagcgc aattctagga gaggattata atagtcaacc 280
<210> 5
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ttaggtcaag tattcgtact caggggtgcc cggctcgagg cgacgggaat caattgccga 60
ttcctccata cgttccagca aagaatccaa tcctgatgct tcactcaagt gaatacccac 120
caacgcagta ccggtctcac ggttgttgcg cttgaggtac tctgccagcg cgatgtcatc 180
atccggtccc aggatatctt ccaggaagtg acgcaactga ccaggctttt gcgggaagtt 240
caccaagaag tagtgcttca aaccgcggtg caccaaggag cgctcagcga tttccgcata 300
acgcagcaca tcgttgttgc caccagagat gatgcacacc acgacagaac caggtgcaaa 360
ggacatttcc ttcaacccag cgatagacag cgcgccagca ggctccgcga tgatgccttc 420
gttttggtaa agatcgagca tctcagtaca cacagcgccc tcggtcgcgc tcatcatgtg 480
cacgcgaccc tggttcttct ccacgatggt gtagttgaga tctccgacac gtttgactgc 540
tgcgccgtcc acaaagggat caacagtctc caaagtgatt ggtctaccat tgtgcaatgc 600
agcctgcatg gatgctgctc ccgctggttc gataccaacg atcgcagtgc gaggtgctat 660
atcagccatg tagctgacca cacctgcaag aagtccgcca ccgccgactg gaaccatcac 720
gtgatctgca ctcttgccca tggaagtcag ctgcgacaag atctcagcag ccactgtacc 780
ctgaccgatg acggtgttgt gagcatcgaa aggctcgatc agcgttgcgc cggtgcgctc 840
tgcatcttca tgcgctgcag ccgatgcttc gtcgaagtta ttgccagtga ccaccaagga 900
gacaaactct ccgccgtgag ccatgatgcg gtcacgcttt tgctttggag tctgcacagg 960
aacatagatg cgtccctgaa cgcccaagga cttgcacaca taggccacgc cctgggcatg 1020
gttacctgca gatgcggcaa cgatacctgc atcgcgctgc tcctgagtga gctgcgctcc 1080
agagttcagc gcaccgcgga tcttgtagga acgaacatcc tgcagatcct cacgcttaag 1140
gtagatttcc gttccggttt cctcagaaag acgagggcaa tactgcaatg gagttggtgc 1200
aatgacggag gaaattcgtg cctgcgccgt ttgaatgtcg gcggcacgaa tcagctccgc 1260
tccgctagcc atcactcctg gacttttctc agacacgtat gtttcactca tggttgacta 1320
ttataatcct ctcctagaat tgcgctcgct gccaggggtt tgtgaaccag gggggcgata 1380
agaacacgtg ctaactcaag cacttaacag cgacgctaca ccaccaaccc tctcctgtac 1440
agacccatca tcacgcgcac cattaagacg caccacacca gcaacttcct gaaccaccgg 1500
ctgtcccaca taccgctgcg ggaactgccg gtaatgggca tccaacgcct gcacacgcaa 1560
ccgatgcacc gccctccaac taccatcaaa c 1591
<210> 6
<211> 253
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tctagagtcg acctgcaggc atgcaagctt aattaattct gtttcctgtg tgaaattgtt 60
atccgctcac aattccacac attatacgag ccgatgatta attgtcaaca gctcatttca 120
gaatatttgc cagaaccgtt atgatgtcgg cgcaaaaaac attatccaga acgggagtgc 180
gccttgagcg acacgaatta tgcagtgatt tacgacctgc acagccatac cacagcttcc 240
gatggctgcc tga 253
<210> 7
<211> 2760
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctagccagag ttgcgcagtg cagtggaaag accgttcatg gtcagctgga tgttgcggga 60
tacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120
gagtggaagc aggtaggggt atcggcgctg gacggatcgt gcgagaagcg ggttgtcatc 180
aagcagatca tcagaaccgg tgattacgca gaacatcttc ttggtcagga agtattcctc 240
gcggatgacg gcataaacgc gctcagctac ttccctatct gggatcaggt ctgcgtagag 300
ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360
gaaaaatggc caggactcgt tgagtgtttg tagctcggca atgcgctggg tggcctgctc 420
cccttcgcca atccattgct caagtgcggt gccgacacca aaccagcccg gcagcatgac 480
acgagactgg gaccaactga gcacccacgg gattgctcgc aaatcttcca ccgaggaggt 540
ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600
ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660
caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720
ggagacgtcg agaagcgatg cctcaagcgt tgctgagacc agagcttcga ggtttcggcg 780
cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840
ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900
gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960
tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020
atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080
gtagttgcgg taaagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140
atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200
ggctgcaatg aggccgaatt ccttgagcaa taccatcggc tcgagcacat cggtgaccga 1260
tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacagcctc 1320
cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380
gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440
ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500
gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560
ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt cattggattc 1620
acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680
agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740
cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800
tggcacgtcg ttgtgcccgg catctgccag cgcaagcagc tgcggggtga ccttattcat 1860
gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920
cacggtttcc gcagcgcggt gagtggaata ctcaactgtt tccgcggtga cataagggtt 1980
accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacatc 2040
ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100
caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160
ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgaccgcgga tgttcttctc 2220
gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280
ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340
agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400
ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460
aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520
caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580
agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640
aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700
aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760
<210> 8
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgggatcctg gcctggatgc acagtggg 28
<210> 9
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctgagtacga atacttgacc taaacatagc tgaaggccac ctcaatc 47
<210> 10
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gattgaggtg gccttcagct atgtttaggt caagtattcg tactcag 47
<210> 11
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
agaggattac actagtcaac cgtttgatgg tagttggagg g 41
<210> 12
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccctccaact accatcaaac ggttgactag tgtaatcctc t 41
<210> 13
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gctctagatg ccaagcagca caagttgt 28
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tagccatcac tcctggactt 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cacacgccag tggttcgaag 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtcgtcggtg cgtcaagcgt 20
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tctagagtcg acctgcaggc caaaccaccg ctaatgatgt 40
<210> 18
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acggcaaagg aacattttcc acgggtactg catatgattg gg 42
<210> 19
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cccaatcata tgcagtaccc gtggaaaatg ttcctttgcc gt 42
<210> 20
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
acaattagaa ggagatcaga gtaatgactg attttttacg cgatgacatc aggttcc 57
<210> 21
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggaacctgat gtcatcgcgt aaaaaatcag tcattactct gatctccttc taattgt 57
<210> 22
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ttgttgcgaa aacaccgaaa ggacgtttga gctgttgaca 40
<210> 23
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgtcaacagc tcaaacgtcc tttcggtgtt ttcgcaacaa 40
<210> 24
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggccagtgcc aagcttgcat ttctgtcata tgcgaagctt 40
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cggtccttga tcaacacctt 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccgtcccttt ttgtcactct 20
<210> 27
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cggaattctc ttcgacgaac tgtgcctt 28
<210> 28
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gtcgactcta gaatgactaa tggagataat ctc 33
<210> 29
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gattatctcc attagtcatt ctagagtcga cctgcaggca 40
<210> 30
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cactaccccc aaatggttca ggcagccatc ggaagctgtg 40
<210> 31
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gatggctgcc tgaaccattt gggggtagtg gccat 35
<210> 32
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
cgggatccga gatctgatca acagagag 28
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
aacttagcgc gaatgatgca 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gtctccaaag acccatccac 20
<210> 35
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
cgggatcctt aagcggtttc tgcgcgag 28
<210> 36
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
cggaattcgg gtgggaattt tcgtgcgttg tg 32
<210> 37
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cgggatccgc acagtgggtt gacgatat 28
<210> 38
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtacgaatac ttgacctaaa catagctgaa ggccacctca 40
<210> 39
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tgaggtggcc ttcagctatg tttaggtcaa gtattcgtac 40
<210> 40
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gaggattaca ctagtcaacc atgagtgaaa catacgtgtc 40
<210> 41
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gacacgtatg tttcactcat ggttgactag tgtaatcctc 40
<210> 42
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gctctagagg tggcgtgttg ccaagcag 28
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
cacacgccag tggttcgaag 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
agccagcagt gtcgtcggtg 20
Claims (10)
1.苏氨酸脱水酶突变体,其特征在于,以谷氨酸棒杆菌野生型苏氨酸脱水酶为参考序列,所述苏氨酸脱水酶突变体含有第54位由丙氨酸突变为除丙氨酸以外的氨基酸、第131位由缬氨酸突变为除缬氨酸以外的氨基酸、第171位由精氨酸突变为除精氨酸以外的氨基酸、第218位由蛋氨酸突变为除蛋氨酸以外的氨基酸、第243位由甘氨酸突变为除甘氨酸以外的氨基酸、第381位由苏氨酸突变为除苏氨酸以外的氨基酸、第383位由苯丙氨酸突变为除苯丙氨酸以外的氨基酸的突变中的任意一种或至少两种的组合。
2.根据权利要求1所述的苏氨酸脱水酶突变体,以谷氨酸棒杆菌野生型苏氨酸脱水酶为参考序列,所述苏氨酸脱水酶突变体含有第54位由丙氨酸突变为苏氨酸、第131位由缬氨酸突变为丙氨酸、第171位由精氨酸突变为组氨酸、第218位由蛋氨酸突变为异亮氨酸、第243位由甘氨酸突变为精氨酸、第381位由苏氨酸突变为丙氨酸、第383位由苯丙氨酸突变为丙氨酸的突变中的任意一种或至少两种的组合;
优选地,所述苏氨酸脱水酶突变体具体如SEQ ID NO.2所示的氨基酸序列。
3.编码权利要求1或2所述的苏氨酸脱水酶突变体的基因。
4.含有权利要求3所述的基因的生物材料,其特征在于,所述生物材料为重组DNA、载体或宿主细胞。
5.一种重组微生物,其特征在于,其表达权利要求1或2所述的苏氨酸脱水酶突变体。
6.根据权利要求5所述的重组微生物,其特征在于,所述苏氨酸脱水酶突变体的编码基因上游可操作性地连接优化的ilvA启动子,所述优化的ilvA启动子为将原始ilvA启动子的-10区突变为TATAAT得到;
优选地,所述优化的ilvA启动子的核苷酸序列如SEQ ID NO.4所示;
所述重组微生物中,在原始ilvA启动子与ilvA编码基因之间插入优化的ilvA启动子。
7.根据权利要求5或6所述的重组微生物,其特征在于,与出发菌株相比,所述重组微生物中,磷酸烯醇式丙酮酸羧化酶的表达和/或酶活性增强;
优选地,与出发菌株相比,所述重组微生物中,6-磷酸葡萄糖脱氢酶的表达和/或酶活性增强;
更优选地,与出发菌株相比,所述重组微生物中,乙酰羟基酸合酶和二羟酸还原异构酶的表达和/或酶活性增强。
8.根据权利要求7所述的重组微生物,其特征在于,表达的增强通过以下任一种方式实现:
(1)增加出发菌株中目标酶编码基因的拷贝数;
(2)将出发菌株中目标酶编码基因的表达调控元件替换为活性更强的调控元件;
(3)优化出发菌株中目标酶编码基因的密码子;
酶活性的增强通过以下任一种方式实现:
(1)将出发菌株中目标酶编码基因的一个或多个碱基进行突变,以使得目标酶的酶活性增强;
(2)将出发菌株中目标酶的编码基因替换为其他生物来源的目标酶编码基因,以使得目标酶的酶活性增强;
优选地,所述重组微生物为棒杆菌属或短杆菌属细菌。
9.权利要求1或2所述的苏氨酸脱水酶突变体或权利要求3所述的基因或权利要求4所述的生物材料或权利要求5~8任一项所述的重组微生物的如下任一种应用:
(1)在工程菌构建中的应用;
(2)在发酵生产支链氨基酸或其衍生物中的应用;
(3)在提高菌株的支链氨基酸或其衍生物的产量和/或转化率中的应用。
10.一种发酵生产支链氨基酸或其衍生物的方法,其特征在于,包括:培养权利要求5~8任一项所述的重组微生物得到培养物,将培养物经分离提取得到支链氨基酸或其衍生物。
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