CN114752544A - 一步法生产γ-氨基丁酸的方法及其菌株构建 - Google Patents
一步法生产γ-氨基丁酸的方法及其菌株构建 Download PDFInfo
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Abstract
本发明公开了一步法生产γ‑氨基丁酸的方法及其菌株构建，属于基因工程和酶工程技术领域。本发明构建了一种可用于γ‑氨基丁酸生产的谷氨酸棒杆菌底盘细胞，并通过表达谷氨酸脱羧酶GAD酶突变体，提高了重组谷氨酸棒杆菌在pH 7.0条件下从葡萄糖一步法发酵生产GABA的产量，使GABA产量达到114 g/L，是目前报道最高值，且极大降低了成本，使GABA生产成本可小于谷氨酸生产成本。
Description
技术领域
本发明涉及一步法生产γ-氨基丁酸的方法及其菌株构建，属于基因工程和酶工程技术领域。
背景技术
γ-氨基丁酸(GABA)是一种四碳的非蛋白氨基酸，它在自然界中广泛存在，从微生物到植物和动物，具有预防及治疗失眠、降压、抗焦虑、镇静、镇痛、利尿等功能，可用于治疗各种神经功能障碍。GABA作为合成2-吡咯烷酮和可生物降解材料-聚酰胺尼龙4的前体也受到了广泛的关注，进一步将其应用领域扩展到工业领域。
GABA的合成有很多方式，包括化学合成，酶促或全细胞生物催化和微生物发酵。与化学合成相比，GABA的生物合成具有许多优势，如简单的后处理程序，更温和的反应条件，较高的产品收率，更高的产物选择性以及更低的环境污染性等。GABA生物合成的主要途径是经L-谷氨酸由谷氨酸脱羧酶GAD催化的不可逆的脱羧反应。谷氨酸脱羧酶（GAD）能够以磷酸吡多醛（PLP）作为辅因子催化将谷氨酸（Glu）生成GABA，是GABA生产的关键性酶。已经有一些国内外的文献报道了关于用谷氨酸棒杆菌生产GABA，在2011年，研究人员首次报道了在谷氨酸棒杆菌中表达来自短乳杆菌Lb85的谷氨酸脱羧酶能够产低浓度但可检测的GABA。另有报道利用外源表达大肠杆菌W3110的GadB，重组谷氨酸棒杆菌经过优化的发酵过程可产生12.37 g/L的GABA。Zhang等人基于重组谷氨酸棒杆菌，从葡萄糖开发有效的直接生物合成途径，无需添加昂贵的PLP辅因子，通过两阶段pH控制策略发酵70小时，GABA产量达到70.6 g/L。
谷氨酸脱羧酶催化谷氨酸生成GABA，该反应消耗质子，从而可以增加环境的pH值。许多谷氨酸脱羧酶在pH4至5范围内均表现出最佳活性，并且其活性在pH值高于6时急剧下降，在pH 7时几乎失活。有些研究报道了一些突变体，提高其在中性pH下活力，但目前研究中，谷氨酸脱羧酶突变体的最适pH都没有改变，大部分没能提高pH高于6时的酶活，在pH7.0条件下仍几乎失活。
目前市场上GABA的生产都是利用谷氨酸作为前体，通过全细胞或者酶催化来实现，而从葡萄糖直接发酵产GABA更具成本优势。生物合成谷氨酸需要在pH 7.0的中性条件下，但GAD最适pH为4至5，在pH 7.0条件下几乎失活。所以即使GABA直接发酵的研究，也一般运用两步法发酵：第一步，中性pH（pH 7左右）条件下产谷氨酸；第二步，酸性pH（pH 5左右）产GABA。与一步法直接发酵生产GABA相比，两步法会导致发酵时间过长，产量较低，成本增加。在常用工业底盘菌株内，与大肠杆菌等相比，谷氨酸棒杆菌（Corynebacterium glutamicum）是安全的生产菌株（General Regarded As Safe, GRAS），其生产的GABA可应用于食品。谷氨酸棒杆菌还是出色的谷氨酸生产菌，而谷氨酸是GABA的直接前体，所以谷氨酸棒杆菌是大规模生产GABA的潜力菌株。总之目前市场中以谷氨酸为底物的全细胞及酶催化，或者两步法发酵，相比较一步法直接发酵，成本都会更高。
发明内容
为解决现有的谷氨酸脱羧酶pH不适用于GABA一步法生产，及全细胞催化，酶催化和两步法生产GABA存在的诸如成本高、产量低、工艺复杂等缺陷，本发明提供了一种在pH7.0条件下具有较高活性的GAD酶突变体及能够利用葡萄糖一步法生产γ-氨基丁酸的谷氨酸棒杆菌。
本发明提供了可用于一步法生产GABA的重组谷氨酸棒杆菌，表达谷氨酸脱羧酶，并进行了如下至少一种改进：
（1）敲除丝氨酸/苏氨酸蛋白激酶PknG；所述丝氨酸/苏氨酸蛋白激酶PknG的Genbank登录号为BAC00145.1；
（2）敲除氨基转移酶bioA，并过表达磷酸烯醇丙酮酸羧化酶（PEPC）和谷氨酸脱氢酶（GDH）；所述氨基转移酶bioA的Genbank登录号为BAB99997.1；所述磷酸烯醇丙酮酸羧化酶（PEPC）的Genbank登录号为BAB98892.1；所述谷氨酸脱氢酶（GDH）的Genbank登录号为BAB99472.1；
（3）敲除转运蛋白（GabP），并过表达丙酮酸羧化酶（PYC）；所述转运蛋白GabP的Genbank登录号为BAB97874.1；所述丙酮酸羧化酶（PYC）的Genbank登录号为BAB98082.1；
（4）敲除磷酸烯醇丙酮酸羧化激酶PCK，并过表达吡哆醛激酶plk；所述磷酸烯醇丙酮酸羧化激酶的Genbank登录号为BAC00257.1，吡哆醛激酶的Genbank登录号为WP_003641112.1；
（5）用启动子PgltA增强柠檬酸合酶基因gltA的表达；所述gltA的核苷酸序列如Genbank登录号：BAB98222.1所示；
（6）敲除草酰乙酸脱羧酶（ODX），将带有弱RBS的酮戊二酸脱氢酶（OdhA）替换到ODX位置，弱RBS序列为CTCACCCACGAGTTCAATAACTAGG，所述ODX的Genbank登录号为BAB98683.1，所述OdhA的Genbank登录号为BAB98522.1；
（7）敲除谷氨酸棒杆菌原有酮戊二酸脱氢酶（OdhA）基因；所述OdhA的Genbank登录号为BAB98522.1；
（8）敲除乳酸脱氢酶（LDH），并过表达异柠檬酸脱氢酶（ICD）；所述乳酸脱氢酶的Genbank登录号为BAC00305.1；所述异柠檬酸脱氢酶的Genbank登录号为BAB98057.1；
（9）敲除乳酸脱氢酶2（LldD），用启动子P tuf 增强谷氨酸脱氢酶（GDH）的表达；所述乳酸脱氢酶2的Genbank登录号为BAC00312.1；所述启动子P tuf 的核苷酸序列如SEQ ID NO.5所示。
在一种实施方式中，所述谷氨酸脱羧酶包括但不限于野生型谷氨酸脱羧酶或谷氨酸脱羧酶突变体。
在一种实施方式中，所述谷氨酸脱羧酶突变体，是在SEQ ID NO.3所示的谷氨酸脱羧酶的基础上，具有如下至少一处突变：
（1）将第38位天冬氨酸突变为天冬酰胺；
（2）将第89位异亮氨酸突变为缬氨酸；
（3）将第92位天冬氨酸突变为天冬酰胺；
（4）将第93位谷氨酸突变为谷氨酰胺；
（5）将第118位天冬氨酸突变为天冬酰胺；
（6）将第153位丝氨酸突变为苏氨酸或丙氨酸；
（7）将第202位天冬氨酸突变为天冬酰胺；
（8）将第268位脯氨酸突变为苏氨酸；
（9）将第294位谷氨酸突变为精氨酸；
（10）将第301位天冬氨酸突变为天冬酰胺；
（11）将第355位苯丙氨酸突变为酪氨酸；
（12）将第371位天冬氨酸突变为天冬酰胺；
（13）将第432位天冬氨酸突变为天冬酰胺；
（14）将第435位组氨酸突变为谷氨酰胺；
（15）将第451位亮氨酸突变为终止密码子；
（16）将第457位赖氨酸突变为终止密码子；
（17）将第461位酪氨酸突变为终止密码子。
在一种实施方式中，所述突变体是将第38位天冬氨酸、第92位天冬氨酸、第118位天冬氨酸、第202位天冬氨酸、第301位天冬氨酸、第371位天冬氨酸、第432位天冬氨酸、第451位亮氨酸、第457位赖氨酸、第461位酪氨酸中的任一个氨基酸进行取代得到的；或者，
将第51位组氨酸、第121位组氨酸、第206位异亮氨酸、第355位苯丙氨酸、第451位亮氨酸、第459位苏氨酸、第461位酪氨酸、第467位组氨酸中的任意两个氨基酸进行取代得到的；或者，
将第68位缬氨酸、第96位谷氨酰胺、第120位苏氨酸、第186位天冬酰胺、第436位亮氨酸、第451位亮氨酸中的任意三个氨基酸进行取代得到的；或者，
将第38位天冬氨酸、第89位异亮氨酸、第92位天冬氨酸、第93位谷氨酸、第153位丝氨酸、第202位天冬氨酸、第268位脯氨酸、第294位谷氨酸、第301位天冬氨酸、第355位苯丙氨酸、第432位天冬氨酸、第435位组氨酸、第451位亮氨酸中的任意四至十三个氨基酸进行取代得到的。
在一种实施方式中，所述突变体将第38位天冬氨酸突变为天冬酰胺，并将第89位异亮氨酸突变为缬氨酸，并将第92位天冬氨酸突变为天冬酰胺，并将第93位谷氨酸突变为谷氨酰胺，并将第153位丝氨酸突变为苏氨酸，并将第202位天冬氨酸突变为天冬酰胺，并将第268位脯氨酸突变为苏氨酸，并将第294位谷氨酸突变为精氨酸，并将第301位天冬氨酸突变为天冬酰胺，并将第355位苯丙氨酸突变为酪氨酸，并将第432位天冬氨酸突变为天冬酰胺，并将第435位组氨酸突变为谷氨酰胺，并将第451位亮氨酸突变为终止密码子（终止密码子用*表示），获得氨基酸序列如SEQ ID NO.4所示的突变体D38N/I89V/D92N//E93Q/S153T/D202N/P268T/E294R/D301N/F355Y/D432N/H435Q/L451*。
在一种实施方式中，编码所述突变体D38N/I89V/D92N//E93Q/S153T/D202N/P268T/E294R/D301N/F355Y/D432N/H435Q/L451*的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中，表达所述谷氨酸脱羧酶的表达载体包括但不限于pCES，pJC1，pAN6质粒，所述质粒pCES公开于论文《Development of a high-copy-number plasmid viaadaptive laboratory evolution of Corynebacterium glutamicum》中，所述质粒pJC1、pAN6公开于论文《Regulation of γ-aminobutyrate (GABA) utilization inCorynebacterium glutamicum by the PucR-type transcriptional regulator GabRand by alternative nitrogen and carbon sources》中。
在一种实施方式中，作为宿主的谷氨酸棒杆菌包括但不限于ATCC 13032、ATCC13869。
本发明还提供了一种一步法生产γ-氨基丁酸的方法，在微生物细胞中，利用碳源一步发酵生产γ-氨基丁酸，发酵过程控制pH在7.0±0.5。
在一种实施方式中，所述具有γ-氨基丁酸合成能力的微生物包括但不限于所述谷氨酸棒杆菌。
在一种实施方式中，所述方法是将所述重组谷氨酸棒杆菌接种至发酵培养基中，发酵至少40 h。
在一种实施方式中，所述发酵培养基以谷氨酸棒杆菌可利用的单糖、多糖或其混合物为碳源，包括但不限于葡萄糖、果糖、蔗糖、糖蜜等。
在一种实施方式中，所述发酵培养基含有：葡萄糖 100g/L, 硫酸铵 12g/L, 硫酸镁 0.87g/L, 玉米浆 3ml/L, 磷酸 0.4ml/L, 氯化钾 0.53g/L, 硫酸亚铁 120mg/L, 硫酸锰 120mg/L, 烟酰胺 42mg/L, 泛酸钙 6.3 mg/L, 维生素B1 6.3mg/L, 生物素0.05mg/L。
在一种实施方式中，发酵过程还控制pH为7.0±0.5。
在一种实施方式中，还控制发酵温度为30℃，溶氧30%。
本发明还提供所述谷氨酸脱羧酶突变体、所述谷氨酸棒杆菌或所述方法在生产含γ-氨基丁酸的产品中的应用。
有益效果：
（1）本发明提供的谷氨酸脱羧酶突变体，极大提高了其pH6.0-7.5条件下的活力，pH7.0时比酶活可达10.29 U/mg。
（2）本发明构建了一种可用于γ-氨基丁酸生产的谷氨酸棒杆菌底盘细胞，并通过表达谷氨酸脱羧酶GAD酶突变体，提高了重组谷氨酸棒杆菌在pH 7.0条件下从葡萄糖一步法发酵生产GABA的产量，使GABA产量达到114 g/L，是目前报道最高值，且极大降低了成本，使GABA生产成本可小于谷氨酸生产成本。
附图说明
图1为GABA的代谢与合成途径以及参与的基因；GAD：谷氨酸脱羧酶，GabT：γ-氨基丁酸氨基转移酶，GabD：琥珀酸半醛脱氢酶，GabP：GABA特异性转运蛋白，ODHC：α-酮戊二酸脱氢酶复合物，PknG：丝氨酸/苏氨酸蛋白激酶，PEPC：磷酸烯醇丙酮酸羧化酶，PC：丙酮酸羧化酶，GDH：谷氨酸脱氢酶。
图2为利用FF10表达谷氨酸脱羧酶突变体（a）或利用FF10表达野生型谷氨酸脱羧酶（b）工程菌一步法发酵生产GABA。
具体实施方式
技术术语：
谷氨酸脱羧酶（glutamate decarboxylase，GAD）：谷氨酸脱羧酶是生物体内正常合成的一种酶蛋白，能够将L-谷氨酸的α-羧基脱去一分子CO2得到γ-氨基丁酸。
表达：术语“表达”包括涉及谷氨酸脱羧酶或谷氨酸脱羧酶突变体产生的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰、以及分泌。
表达载体：术语“表达载体”意指直链或环状DNA分子，所述分子包含编码本发明的谷氨酸脱羧酶突变体的多核苷酸并且可操作地连接至提供用于其表达的控制序列。
宿主细胞：术语“宿主细胞”意指易于用包含本发明的多核苷酸的核酸构建体或表达载体进行转化、转染、转导等的任何细胞类型。术语“宿主细胞”涵盖由于复制期间出现的突变而与亲本细胞不完全相同的任何亲本细胞子代。
突变体：意指具有谷氨酸脱羧酶活性的、在一个或多个(例如若干个)位置包含改变(即取代、插入和/或缺失)的多肽。取代意指用不同的氨基酸替代占据某一位置的氨基酸；缺失意指去除占据某一位置的氨基酸；而插入意指在邻接并且紧随占据某一位置的氨基酸之后添加氨基酸。本发明的突变体母本具有SEQ ID NO.3所示的氨基酸序列，并在第38位、第89位、第92位、第93位、第153位、第202位、第268位、第294位、第301位、第355位、第371位、第432位、第435位、第451位、第461位中的至少一处发生取代；在此基础上，还可同时发生第38位、第89位、第92位、第93位、第153位、第202位、第268位、第294位、第301位、第355位、第432位、第435位、第451位的取代。本发明的谷氨酸脱羧酶突变体的活性为母本谷氨酸脱羧酶活性的至少20％，例如至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少100％。
野生型谷氨酸脱羧酶：术语“野生型”谷氨酸脱羧酶意指由见于自然界中的天然存在的微生物(如细菌、酵母或丝状真菌)表达的谷氨酸脱羧酶。
突变：对于氨基酸取代，使用以下命名法：原始氨基酸、位置、被取代的氨基酸。例如，将在位置38处的天冬氨酸被天冬酰胺取代表示为“Asp38Asn”或“D38N”。多个突变通过符号(“/”)分开，例如“D38N/I89V/D92N//E93Q/S153T/D202N/P268T/E294R/D301N/F355Y/D432N/H435Q/L451*”代表在位置38处的天冬氨酸被天冬酰胺取代，在位置89处的异亮氨酸被缬氨酸取代，在位置92处的天冬氨酸被天冬酰胺取代，在位置93处的谷氨酸被谷氨酰胺，在位置153处的丝氨酸被苏氨酸取代，在位置202处的天冬氨酸被天冬酰胺取代，在位置268处的脯氨酸被苏氨酸取代，在位置294处的谷氨酸被精氨酸取代，在位置301处的天冬氨酸被天冬酰胺取代，在位置355处的苯丙氨酸被酪氨酸取代，在位置432处的天冬氨酸被天冬酰胺取代，在位置435处的组氨酸被谷氨酰胺取代，在位置451处的亮氨酸被终止密码子取代。
发酵液：“发酵液”是指由细胞发酵产生的、未经回收或经过回收和/或纯化的制剂。例如，当微生物培养物在允许蛋白质合成(例如，由宿主细胞表达酶)并且将蛋白质分泌到细胞培养基中的碳限制条件下孵育生长到饱和时，产生发酵液。所述发酵液可以含有在发酵结束时得到的发酵材料的内容物。例如，所述发酵液包含被微生物利用后的培养基成分以及可通过离心去除微生物细胞(例如，丝状真菌细胞)后存在的细胞碎片。
培养基：
CGXII培养基：葡萄糖 50g/L，(NH4)2SO4 20g/L，尿素 5g/L，KH2PO4 1g/L，K2HPO41g/L，MgSO4·7H2O 0.25g/L，CaCl2·2H2O 13.3mg/L，MOPS 42g/L，生物素 0.2mg/L，微量元素溶液 1ml/L，运用KOH 将pH 调节到 7.0；其中，微量元素溶液：FeSO4·7H2O 10g/L，MnSO4·1H2O 10g/L，ZnSO4·7H2O 1g/L，CuSO4·5H2O 313mg/L，NiCl·6H2O 20mg/L。
发酵培养基：葡萄糖 100g/L, 硫酸铵 12g/L, 硫酸镁 0.87g/L, 玉米浆 3ml/L,磷酸 0.4ml/L, 氯化钾 0.53g/L, 硫酸亚铁 120mg/L, 硫酸锰 120mg/L, 烟酰胺 42mg/L, 泛酸钙 6.3 mg/L, 维生素B1 6.3mg/L, 生物素 0.5mg/L。
检测方法：
谷氨酸脱羧酶的测定：通过高效液相法检测酶反应体系或发酵液中的GABA含量（方法参照2020年公开的论文《Regulation of γ-aminobutyrate (GABA) utilizationin Corynebacterium glutamicum by the PucR-type transcriptional regulator GabRand by alternative nitrogen and carbon sources》），以谷氨酸为底物，PLP作为辅酶，在pH 7.0条件下测定GAD活性。通过添加酶开始反应，添加氢氧化钠结束反应，检测相应GABA的产量。根据GABA含量计算谷氨酸脱羧酶酶活。每分钟生成1微摩尔GABA所需的酶量定义为一个酶活单位（U）。比酶活表示为每毫克蛋白质有多少U谷氨酸脱羧酶。
实施例1 产γ-氨基丁酸的谷氨酸棒杆菌工程菌株底盘细胞的构建
图1为GABA的合成途径和代谢途径，敲除其代谢途径和一些支路途径，增强合成途径中一些关键基因表达，构建能够合成γ-氨基丁酸的谷氨酸棒杆菌，具体如下：
（1）构建重组质粒pK18-ΔpknG，敲除丝氨酸/苏氨酸蛋白激酶 PknG，进一步降低α-酮戊二酸脱氢酶复合物（ODHC）的活性：以谷氨酸棒杆菌基因组为模板分别克隆pknG基因上下游1000 bp的同源臂，与Pk18mobsacB骨架通过Gibson方法相连，将获得的重组质粒pK18-ΔpknG转化至谷氨酸棒杆菌ATCC 13032中，重组得到菌株FF1/ΔpknG，命名为FF2。
（2）敲除氨基转移酶BioA，同时增强磷酸烯醇丙酮酸羧化酶（PEPC）和谷氨酸脱氢酶（GDH）的表达：以谷氨酸棒杆菌基因组为模板分别克隆bioA基因（Genbank登录号BAB99997.1）上下游1000 bp的同源臂，克隆含启动子的磷酸烯醇丙酮酸羧化酶（Genbank登录号BAB98892.1）的编码基因pepc与含启动子的谷氨酸脱氢酶（Genbankd登录号：BAB99472.1）基因gdh，构建在bioA基因上下游同源臂之间，与Pk18mobsacB骨架通过Gibson方法相连，构建重组质粒pK18-ΔbioA::pepc+gdh，将重组质粒pK18-ΔbioA::pepc+gdh转化至菌株FF2感受态细胞中，重组得到菌株FF2/ΔbioA::pepc+gdh，命名为FF3。
（3）按照上述相同策略构建重组质粒pK18-ΔgabP::pyc，敲除GABA往胞里转运的蛋白GabP（Genbank登录号：BAB97874.1），并增强丙酮酸羧化酶PYC（Genbank登录号：BAB98082.1）的表达，将构建的重组质粒pK18-ΔgabP::pyc转化至FF3感受态细胞中，重组得到菌株FF3/ΔgabP::pyc，命名为FF4。
（4）按照上述相同策略构建重组质粒pK18-Δpck::plk，敲除磷酸烯醇丙酮酸羧化激酶PCK（Genbank登录号为BAC00257.1），并过表达吡哆醛激酶plk（Genbank登录号：WP_003641112.1）增强辅酶PLP的合成，将构建的重组质粒pK18-Δpck::plk转化至FF4感受态细胞中，得到重组菌株FF4/Δpck::plk，命名为FF5。
（5）按照上述相同策略构建重组质粒pK18-PgltA gltA，用SEQ ID NO.6所示的启动子PgltA增强增强柠檬酸合酶GltA（Genbank登录号：BAB98222.1）的表达，将构建的重组质粒pK18-PgltA gltA转化至FF5感受态细胞中，重组得到菌株FF5 PgltA gltA，命名为FF6。
（6）按照上述相同策略构建重组质粒pK18-Δodx::wRBSodhA，敲除草酰乙酸脱羧酶ODX（Genbank登录号：BAB98683.1），并将带有弱RBS的酮戊二酸脱氢酶基因OdhA替换到ODX所在位置，弱RBS序列为CTCACCCACGAGTTCAATAACTAGG；将重组质粒pK18-Δodx::wRBSodhA转化至FF6感受态细胞中，重组得到菌株FF6 Δodx::wRBSodhA，命名为FF7。
（7）按照上述相同策略构建重组质粒pK18-ΔodhA，敲除谷氨酸棒杆菌原有的酮戊二酸脱氢酶OdhA（Genbank登录号为BAB98522.1）,将重组质粒pK18-ΔodhA转化至FF7感受态细胞中，重组得到菌株FF7 ΔodhA，命名为FF8。
（8）照上述相同策略构建重组质粒pK18-Δldh::icd，敲除乳酸脱氢酶LDH（Genbank登录号：BAC00305.1），增强异柠檬酸脱氢酶ICD（Genbank登录号：BAB98057.1）的表达，将重组质粒pK18-Δldh::icd转化至FF8感受态细胞中，重组得到菌株FF8 Δldh:: icd，命名为FF9。
（9）照上述相同策略构建重组质粒pK18-Δlldd::P tuf gdh，敲除乳酸脱氢酶2 LldD（Genbank登录号：BAC00312.1），用SEQ ID NO.5所示的启动子P tuf 增强谷氨酸脱氢酶GDH（Genbank登录号：BAB99472.1）的表达，将重组质粒pK18-Δlldd::P tuf gdh转化至FF9感受态细胞中，重组得到菌株FF9 Δlldd::P tuf gdh，命名为FF10。
实施例2 谷氨酸脱羧酶突变体、重组质粒和重组菌的构建
设计扩增引物：
GADF: CTTGGTTGGTAGGAGTAGCATGGGATCCATGCCTCAATGGCATCCGCATCGTGA，
GADR:CTACTGCCGCCAGGCAGCGGCCGCTTAATGATGAAATCCATTGTCCTATTTC，
以巨大芽孢杆菌(Bacillus magaterium)CICC 10055基因组为模板，通过易错PCR进行约25轮的扩增，获得SEQ ID NO.1所示野生型谷氨酸脱羧酶基因的随机突变库，将扩增产物经DNA纯化试剂盒纯化后，通过Gibson将易错PCR扩增产物和质粒pCES（质粒公开于论文《Development of a high-copy-number plasmid via adaptive laboratoryevolution of Corynebacterium glutamicum》）的主干片段相连，转化至E.coli DH5α中，构建突变库。将突变菌株在适合GABA生产的CGXII培养基中，在30℃培养30小时后，依靠高效液相HPLC检测GABA产量，确认酶活增加的突变株。再对突变株的基因序列进行测序分析，得到GABA的产量以及相应突变位点的关联信息。
经筛选，获得了具有如下至少一处突变的突变体库：第38位天冬氨酸突变为天冬酰胺、第89位异亮氨酸突变为缬氨酸、第92位天冬氨酸突变为天冬酰胺、第93位谷氨酸突变为谷氨酰胺、第118位天冬氨酸突变为天冬酰胺、第153位丝氨酸突变为苏氨酸或丙氨酸、第202位天冬氨酸突变为天冬酰胺、第268位脯氨酸突变为苏氨酸、第294位谷氨酸突变为精氨酸、第301位天冬氨酸突变为天冬酰胺、第355位苯丙氨酸突变为酪氨酸、第371位天冬氨酸突变为天冬酰胺、第432位天冬氨酸突变为天冬酰胺、第435位组氨酸突变为谷氨酰胺、第451位亮氨酸突变为终止密码子、第457位赖氨酸突变为终止密码子、第461位酪氨酸突变为终止密码子。
其中，氨基酸序列如SEQ ID NO,4所示的突变体，在野生型谷氨酸脱羧酶的基础上具有：D38N/I89V/D92N/E93Q/S153T/D202N/P268T/E294R/D301N/F355Y/D432N/H435Q/L451*所示突变，命名为GAD MUT。
将GAD突变体GAD MUT构建到pET质粒中，并转化进大肠杆菌BL21（DE3）后，在LB培养基中于37°C振荡培养。当OD600达到 0.6~0.8 时，加入终浓度0.2 mM异丙基-β-D-硫代吡喃半乳糖苷 (IPTG)诱导基因表达。在25℃诱导8小时后，通过离心收获细胞。将收获的细胞重新悬浮在结合缓冲液（20 mM Tris-HCl [pH 7.8]、500 mM氯化钠和10 mM咪唑）中，接着通过超声破碎。收集破碎后的上清液，通过镍亲和层析来纯化GAD。纯化后的蛋白利用HisTrap HP 5-ml 脱盐柱进行脱盐。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测蛋白纯化质量。以牛血清白蛋白为标准，采用 Bradford 法测定蛋白质浓度。纯化后，检测突变体GAD MUT的比酶活，以野生型谷氨酸脱羧酶(GAD WT)为对照。结果显示，谷氨酸脱羧酶野生型（GAD WT）在pH 7.0条件下比酶活是0.84 U/mg，而本发明构建的谷氨酸脱羧酶突变体D38N/I89V/D92N/E93Q/S153T/D202N/P268T/E294R/D301N/F355Y/D432N/H435Q/L451*（GAD MUT）在pH 7.0条件下的比酶活是10.29 U/mg，与野生型相比提高了12.25倍。
实施例3 利用谷氨酸棒杆菌工程菌株发酵生产γ-氨基丁酸
将按照实施例2的策略得到的携带突变体GAD MUT编码序列的表达载体pCES-GADMUT转化到实施例1构建的谷氨酸棒杆菌工程菌株FF10中，用于一步法从葡萄糖发酵生产γ-氨基丁酸。以在菌株FF10中表达谷氨酸脱羧酶野生型（GAD WT）的菌株为对照。
将菌株FF10 pCES-GAD MUT在BHIS培养基中，于30℃培养24小时，获得种子液；将500 mL发酵培养基加到1L发酵罐中，按10%的接种率将种子液接种到发酵罐，发酵温度为30℃，溶氧30%，利用氨水来调节pH 7.0±0.5。如图2所示，以FF10为宿主表达谷氨酸脱羧酶突变体（GAD MUT）的菌株发酵168小时后，GABA产量可达114 g/L。
以在FF10中表达谷氨酸脱羧酶野生型（GAD WT）的菌株为对照，采用两步法发酵，发酵过程初期采用氨水调节pH 7.0±0.5，生产谷氨酸，随着发酵的进行，76小时后pH控制为5.5，至168 h可发酵生产27 g/L GABA。
本发明构建的重组谷氨酸棒杆菌能够在pH7.0的条件下实现一步法发酵，且产量是对照菌株两步法发酵结果的4.2倍多，是目前报道的最高值。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 森瑞斯生物科技（深圳）有限公司
中国科学院深圳先进技术研究院
<120> 一步法生产γ-氨基丁酸的方法及其菌株构建
<130> IBAA220364A
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1404
<212> DNA
<213> Bacillus magaterium
<400> 1
atgcctcaat ggcatccgca tcgtgaacaa aaaaatttac ctgatgaatt tcctgttaat 60
ccgctttttt ctcgacaagg agaagtgaca attccaagac tgcgtatcgg tgatcaaggt 120
atgcttccgg aaacggctta tcaaatcatt catgacgaaa ttgctttaga cggaaatgcc 180
cgcttgaatt tagctacgtt tgttactacg tggatggagc ctgatgcaaa gcgtttgtac 240
ggagaatctt ttgataaaaa tatgatcgat aaagatgagt atccgcagac agcggctatt 300
gaagagagat gtgtacgtat tttagcggat ttgtggaatt cacctaatcc tgataccacg 360
atgggcgttt ctactacagg ttcatctgaa gcatgtatgc ttggtggact agcgttaaaa 420
agacgatggc agaaactgcg taaaagtaaa gggctatcaa cggaccgccc caatattgta 480
tttagttcat cggttcaagt ggtatgggag aagttcgcaa actattggga cgtagagcct 540
cgttatgtga atattaatcc agatcatcct tatttagatg cagaaggcgt gattaatgcg 600
gttgacgaaa atacaattgg cgtcgtaccg attcttggag tcacgtatac agggggttac 660
gaaccaatag ctgctatcgc aaaagcatta gatgagttac aggaaaaaac agggttggat 720
attcctatcc atgtagatgc tgcttctgga ggttttatcg ctccatttct tcaaccagac 780
cttatctggg atttccgctt gccgcgagta aagtccatta acgtgtcagg acacaagtat 840
ggtttagttt accctggctt gggatgggtg atttggagag aaaaagagga cttgcctgaa 900
gatcttattt tccgcgtttc ttatttaggg ggcaacatgc caacttttgc gctcaacttc 960
tctagaccag gagcacaagt ccttttgcag tactacaatt tcttgcgttt aggtaaagac 1020
ggctattatg ccgtgcaaaa aacctcccaa gaaaacgcgc tgtttcttag caaagaaatt 1080
ggagaaatgg acgcattcga aattcttgct gatggttcag atatcccggt tcttgcttgg 1140
aaactgaaag aagactatac accaaactgg actctttatg atttgtctag acaactgcgt 1200
acgtacggat ggcaagttcc tgcttaccca ctcccagcag acatggaaga aatcacaatc 1260
atgcgcattg ttgttagaaa tgggttttca agagaccttg ctcatttatt tatggttaat 1320
ttcaaacaag ccgttgaatt tcttaactcg ttggatagac ctgttcttaa agacacgaaa 1380
tacgacaatg gatttcatca ttaa 1404
<210> 2
<211> 1353
<212> DNA
<213> 人工序列
<400> 2
atgcctcaat ggcatccgca tcgtgaacaa aaaaatttgc ctgatgaatt tcctgttaat 60
ccgctttttt ctcgacaagg agaagtgaca attccaagac tgcgtatcgg taatcaaggt 120
atgcttccgg aaacggctta tcaaatcatt catgacgaaa ttgctttaga cggaaatgcc 180
cgcttgaatt tagctacgtt tgttactacg tggatggagc ctgatgcaaa gcgtttgtac 240
ggagaatctt ttgataaaaa tatggtcgat aaaaatcagt atccgcagac agcggctatt 300
gaagagagat gtgtacgtat tttagcggat ttgtggaatt cacctaatcc tgataccacg 360
atgggcgttt ctactacagg ttcatctgaa gcatgtatgc ttggtggact agcgttaaaa 420
agacgatggc agaaactgcg taaaagtaaa gggctaacaa cggaccgccc caatattgta 480
tttagttcat cggttcaagt ggtatgggag aagttcgcaa actattggga cgtagagcct 540
cgttatgtga atattaatcc agatcatcct tatttagatg cagaaggcgt gattaatgcg 600
gttaatgaaa atacaattgg cgtcgtaccg attcttggag tcacgtatac agggggttac 660
gaaccaatag ctgctatcgc aaaagcatta gatgagttac aggaaaaaac agggttggat 720
attcctatcc atgtggatgc tgcttctgga ggttttatcg ctccatttct tcaaccagac 780
cttatctggg atttccgctt gacgcgagta aagtccatta acgtgtcagg acacaagtat 840
ggtttagttt accctggctt gggatgggtg atttggagaa gaaaagagga cttgcctgaa 900
aatcttattt tccgcgtttc ttatttaggg ggcaacatgc caacttttgc gctcaacttc 960
tctagaccag gagcacaagt ccttttgcag tactacaatt tcttgcgttt aggtaaagac 1020
ggctattatg ccgtgcaaaa aacctcccaa gaaaacgcgc tgtatcttag caaagaaatt 1080
ggagaaatgg acgcattcga aattcttgct gatggttcag atatcccggt tcttgcttgg 1140
aaactgaaag aagactatac accaaactgg actctttatg atttgtctag acaactgcgt 1200
acgtacggat ggcaagttcc agcttaccca ctcccagcag acatggaaga aatcacaatc 1260
atgcgcattg ttgttagaaa tgggttttca agaaaccttg ctcaattatt tatggttaat 1320
ttcaaacaag ccgttgaatt tcttaactcg tag 1353
<210> 3
<211> 467
<212> PRT
<213> Bacillus magaterium
<400> 3
Met Pro Gln Trp His Pro His Arg Glu Gln Lys Asn Leu Pro Asp Glu
1 5 10 15
Phe Pro Val Asn Pro Leu Phe Ser Arg Gln Gly Glu Val Thr Ile Pro
20 25 30
Arg Leu Arg Ile Gly Asp Gln Gly Met Leu Pro Glu Thr Ala Tyr Gln
35 40 45
Ile Ile His Asp Glu Ile Ala Leu Asp Gly Asn Ala Arg Leu Asn Leu
50 55 60
Ala Thr Phe Val Thr Thr Trp Met Glu Pro Asp Ala Lys Arg Leu Tyr
65 70 75 80
Gly Glu Ser Phe Asp Lys Asn Met Ile Asp Lys Asp Glu Tyr Pro Gln
85 90 95
Thr Ala Ala Ile Glu Glu Arg Cys Val Arg Ile Leu Ala Asp Leu Trp
100 105 110
Asn Ser Pro Asn Pro Asp Thr Thr Met Gly Val Ser Thr Thr Gly Ser
115 120 125
Ser Glu Ala Cys Met Leu Gly Gly Leu Ala Leu Lys Arg Arg Trp Gln
130 135 140
Lys Leu Arg Lys Ser Lys Gly Leu Ser Thr Asp Arg Pro Asn Ile Val
145 150 155 160
Phe Ser Ser Ser Val Gln Val Val Trp Glu Lys Phe Ala Asn Tyr Trp
165 170 175
Asp Val Glu Pro Arg Tyr Val Asn Ile Asn Pro Asp His Pro Tyr Leu
180 185 190
Asp Ala Glu Gly Val Ile Asn Ala Val Asp Glu Asn Thr Ile Gly Val
195 200 205
Val Pro Ile Leu Gly Val Thr Tyr Thr Gly Gly Tyr Glu Pro Ile Ala
210 215 220
Ala Ile Ala Lys Ala Leu Asp Glu Leu Gln Glu Lys Thr Gly Leu Asp
225 230 235 240
Ile Pro Ile His Val Asp Ala Ala Ser Gly Gly Phe Ile Ala Pro Phe
245 250 255
Leu Gln Pro Asp Leu Ile Trp Asp Phe Arg Leu Pro Arg Val Lys Ser
260 265 270
Ile Asn Val Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Leu Gly
275 280 285
Trp Val Ile Trp Arg Glu Lys Glu Asp Leu Pro Glu Asp Leu Ile Phe
290 295 300
Arg Val Ser Tyr Leu Gly Gly Asn Met Pro Thr Phe Ala Leu Asn Phe
305 310 315 320
Ser Arg Pro Gly Ala Gln Val Leu Leu Gln Tyr Tyr Asn Phe Leu Arg
325 330 335
Leu Gly Lys Asp Gly Tyr Tyr Ala Val Gln Lys Thr Ser Gln Glu Asn
340 345 350
Ala Leu Phe Leu Ser Lys Glu Ile Gly Glu Met Asp Ala Phe Glu Ile
355 360 365
Leu Ala Asp Gly Ser Asp Ile Pro Val Leu Ala Trp Lys Leu Lys Glu
370 375 380
Asp Tyr Thr Pro Asn Trp Thr Leu Tyr Asp Leu Ser Arg Gln Leu Arg
385 390 395 400
Thr Tyr Gly Trp Gln Val Pro Ala Tyr Pro Leu Pro Ala Asp Met Glu
405 410 415
Glu Ile Thr Ile Met Arg Ile Val Val Arg Asn Gly Phe Ser Arg Asp
420 425 430
Leu Ala His Leu Phe Met Val Asn Phe Lys Gln Ala Val Glu Phe Leu
435 440 445
Asn Ser Leu Asp Arg Pro Val Leu Lys Asp Thr Lys Tyr Asp Asn Gly
450 455 460
Phe His His
465
<210> 4
<211> 450
<212> PRT
<213> 人工序列
<400> 4
Met Pro Gln Trp His Pro His Arg Glu Gln Lys Asn Leu Pro Asp Glu
1 5 10 15
Phe Pro Val Asn Pro Leu Phe Ser Arg Gln Gly Glu Val Thr Ile Pro
20 25 30
Arg Leu Arg Ile Gly Asn Gln Gly Met Leu Pro Glu Thr Ala Tyr Gln
35 40 45
Ile Ile His Asp Glu Ile Ala Leu Asp Gly Asn Ala Arg Leu Asn Leu
50 55 60
Ala Thr Phe Val Thr Thr Trp Met Glu Pro Asp Ala Lys Arg Leu Tyr
65 70 75 80
Gly Glu Ser Phe Asp Lys Asn Met Val Asp Lys Asn Gln Tyr Pro Gln
85 90 95
Thr Ala Ala Ile Glu Glu Arg Cys Val Arg Ile Leu Ala Asp Leu Trp
100 105 110
Asn Ser Pro Asn Pro Asp Thr Thr Met Gly Val Ser Thr Thr Gly Ser
115 120 125
Ser Glu Ala Cys Met Leu Gly Gly Leu Ala Leu Lys Arg Arg Trp Gln
130 135 140
Lys Leu Arg Lys Ser Lys Gly Leu Thr Thr Asp Arg Pro Asn Ile Val
145 150 155 160
Phe Ser Ser Ser Val Gln Val Val Trp Glu Lys Phe Ala Asn Tyr Trp
165 170 175
Asp Val Glu Pro Arg Tyr Val Asn Ile Asn Pro Asp His Pro Tyr Leu
180 185 190
Asp Ala Glu Gly Val Ile Asn Ala Val Asn Glu Asn Thr Ile Gly Val
195 200 205
Val Pro Ile Leu Gly Val Thr Tyr Thr Gly Gly Tyr Glu Pro Ile Ala
210 215 220
Ala Ile Ala Lys Ala Leu Asp Glu Leu Gln Glu Lys Thr Gly Leu Asp
225 230 235 240
Ile Pro Ile His Val Asp Ala Ala Ser Gly Gly Phe Ile Ala Pro Phe
245 250 255
Leu Gln Pro Asp Leu Ile Trp Asp Phe Arg Leu Thr Arg Val Lys Ser
260 265 270
Ile Asn Val Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Leu Gly
275 280 285
Trp Val Ile Trp Arg Arg Lys Glu Asp Leu Pro Glu Asn Leu Ile Phe
290 295 300
Arg Val Ser Tyr Leu Gly Gly Asn Met Pro Thr Phe Ala Leu Asn Phe
305 310 315 320
Ser Arg Pro Gly Ala Gln Val Leu Leu Gln Tyr Tyr Asn Phe Leu Arg
325 330 335
Leu Gly Lys Asp Gly Tyr Tyr Ala Val Gln Lys Thr Ser Gln Glu Asn
340 345 350
Ala Leu Tyr Leu Ser Lys Glu Ile Gly Glu Met Asp Ala Phe Glu Ile
355 360 365
Leu Ala Asp Gly Ser Asp Ile Pro Val Leu Ala Trp Lys Leu Lys Glu
370 375 380
Asp Tyr Thr Pro Asn Trp Thr Leu Tyr Asp Leu Ser Arg Gln Leu Arg
385 390 395 400
Thr Tyr Gly Trp Gln Val Pro Ala Tyr Pro Leu Pro Ala Asp Met Glu
405 410 415
Glu Ile Thr Ile Met Arg Ile Val Val Arg Asn Gly Phe Ser Arg Asn
420 425 430
Leu Ala Gln Leu Phe Met Val Asn Phe Lys Gln Ala Val Glu Phe Leu
435 440 445
Asn Ser
450
<210> 5
<211> 400
<212> DNA
<213> 人工序列
<400> 5
cagatgttat tgctgagcgc aacggcaccg cttcctaaag atcgtttaga tccgaaggaa 60
aacgtcgaaa agcaatttgc ttttcgacgc cccaccccgc gcgttttagc gtgtcagtag 120
gcgcgtaggg taagtggggt agcggcttgt tagatatctt gaaatcggct ttcaacagca 180
ttgatttcga tgtatttagc tggccgttac cctgcgaatg tccacagggt agctggtagt 240
ttgaaaatca acgccgttgc ccttaggatt cagtaactgg cacattttgt aatgcgctag 300
atctgtgtgc tcagtcttcc aggctgctga tcacagtgaa agcaaaacca attcgtggct 360
gcgaaagtcg tagccaccac gaagtccagg aggacataca 400
<210> 6
<211> 498
<212> DNA
<213> 人工序列
<400> 6
caatttctag gttgttaata tcccctgagg ttgcgttata gggtggcgaa ttgcatgggg 60
aaagctactc ggcacccatc cttgtcgcgt gcatcacaaa ctttgctaaa ctgtgcacca 120
gtccacttat tgtgggattt ttaatgcctt aaaggccagc attttcaccc tctagcgggg 180
ttgaatgctg gccttgaggg tgcagaacta aatagcagca catcggcaca attgatctga 240
gttctattgg cgtgaccgtg gctactgatt acggtggctg tgggtggtcg ggaatgatgt 300
aaccaacgtg attgtggggg aattggctct cacttcggat atggctaaac cgcatttatc 360
ggtatagcgt gttaaccgga ccagattggg aaagaaatgt gtcgagtaac aaaaactgac 420
atgcgcttgg cgcatcccag ttggtaagaa taaacgggac tacttccgta atccggaaga 480
gtttttttcc gaacaaat 498
Claims (10)
1.一种重组谷氨酸棒杆菌，其特征在于，表达谷氨酸脱羧酶，并进行了（1）~（9）的改进：
（1）敲除或缺失丝氨酸/苏氨酸蛋白激酶基因pknG；
（2）敲除或缺失氨基转移酶基因bioA，并过表达磷酸烯醇丙酮酸羧化酶和谷氨酸脱氢酶；
（3）敲除或缺失转运蛋白基因gabP，并过表达丙酮酸羧化酶；
（4）敲除或缺失磷酸烯醇丙酮酸羧化激酶基因pck，并过表达吡哆醛激酶；
（5）用启动子P gltA 增强柠檬酸合酶基因gltA的表达；
（6）敲除或缺失草酰乙酸脱羧酶基因odx；
（7）用弱RBS表达酮戊二酸脱氢酶基因odhA；
（8）敲除或缺失乳酸脱氢酶基因ldh，并过表达异柠檬酸脱氢酶；
（9）敲除或缺失乳酸脱氢酶2基因lldD，用启动子P tuf 增强谷氨酸脱氢酶基因gdh的表达。
2.根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，所述谷氨酸脱羧酶具有SEQID NO.4所示的氨基酸序列。
3.根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，所述谷氨酸脱羧酶在SEQ IDNO.3的基础上，具有：第38位、51位，68位、89位、92位、93位、96位、118位、120位、121位、153位、186位、202位、206位、268位、294位、301位、355位、371位、432位、436位、451位、457位、459位、461位或467位中至少一处氨基酸的突变。
4.根据权利要求1~3任一所述的重组谷氨酸棒杆菌，其特征在于，以pCES、pJC1或pAN6质粒为表达载体表达所述谷氨酸脱羧酶。
5.根据权利要求1~3任一所述的重组谷氨酸棒杆菌，其特征在于，以谷氨酸棒杆菌ATCC13032或谷氨酸棒杆菌ATCC 13869为宿主细胞。
6.一种一步法生产γ-氨基丁酸的方法，其特征在于，不添加谷氨酸或谷氨酸盐作为生产前体，不用生长后再收集菌体做转化，直接从包括葡萄糖在内的碳源一步发酵生产γ-氨基丁酸，发酵过程控制pH在7.0±0.5。
7.根据权利要求6所述的方法，其特征在于，以权利要求1~5任一所述的重组谷氨酸棒杆菌发酵，控制发酵温度为28~30℃，发酵至少40 h。
8.根据权利要求6或7所述的方法，其特征在于，所述碳源为单糖、多糖或其混合物。
9.根据权利要求6或7所述的方法，其特征在于，用于发酵的培养基含有：葡萄糖、硫酸铵、硫酸镁、玉米浆、磷酸、氯化钾、硫酸亚铁、硫酸锰、烟酰胺、泛酸钙、维生素B1、生物素。
10.权利要求1~5任一所述的重组谷氨酸棒杆菌，或权利要求6~9任一所述方法在生产含γ-氨基丁酸的产品中的应用。
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| CN115851566A (zh) * | 2022-12-02 | 2023-03-28 | 森瑞斯生物科技(深圳)有限公司 | 一步法生产2-吡咯烷酮的菌株及应用 |
| CN116555138A (zh) * | 2023-02-06 | 2023-08-08 | 潍坊亚森生物科技有限公司 | 乙酰辅酶a合成酶acs突变体及其在2-吡咯烷酮生产中的应用 |
| CN117165504A (zh) * | 2023-08-03 | 2023-12-05 | 天津世纪伟康生物科技有限公司 | 一种发酵法高效生产γ-氨基丁酸的工程菌及其应用 |
| WO2023240871A1 (zh) * | 2022-06-16 | 2023-12-21 | 森瑞斯生物科技(深圳)有限公司 | 谷氨酸脱羧酶突变体及在生产γ-氨基丁酸中的应用 |
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| CN116555138A (zh) * | 2023-02-06 | 2023-08-08 | 潍坊亚森生物科技有限公司 | 乙酰辅酶a合成酶acs突变体及其在2-吡咯烷酮生产中的应用 |
| CN116555138B (zh) * | 2023-02-06 | 2024-02-13 | 森瑞斯生物科技(深圳)有限公司 | 乙酰辅酶a合成酶acs突变体及其在2-吡咯烷酮生产中的应用 |
| CN117165504A (zh) * | 2023-08-03 | 2023-12-05 | 天津世纪伟康生物科技有限公司 | 一种发酵法高效生产γ-氨基丁酸的工程菌及其应用 |
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