CN117126792A - 一种用于生产l-茶氨酸的重组质粒、基因工程菌株及方法 - Google Patents
一种用于生产l-茶氨酸的重组质粒、基因工程菌株及方法 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,具体涉及一种生产L‑茶氨酸的重组质粒、基因工程菌株及其发酵应用方法。本发明构建一种基于多聚磷酸盐激酶PPK的ATP再生系统,同时过表达谷氨酰胺转运蛋白GNP1强化L‑茶氨酸的转运效率。本发明提供的γ‑谷氨酰甲胺合成酶突变体,其在氨基酸序列如SEQ ID NO:1所示的基础上,在选自下组的一个或两个氨基酸残基位点发生突变:174位和247位。所述突变体可以提高菌株L‑茶氨酸的产量。本发明利用廉价的葡萄糖为底物,不需要额外添加乙胺,建立L‑茶氨酸生物转化系统,提供一种用于生产L‑茶氨酸的低成本,高效转化率的微生物发酵方法,发酵48h,L‑茶氨酸的产量达到76.4g/L。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种生产L-茶氨酸的重组质粒、基因工程菌株及其发酵应用方法。
背景技术
L-茶氨酸是茶叶中特征氨基酸,占其总游离氨基酸40%以上。由于L-茶氨酸具有多种保健功能,如降血压、预防血管疾病、抗肿瘤、缓解压力、保护神经、减肥降脂和提高免疫系统能力,已被商业开发为一种有价值的成分,被广泛用与食品、制药、保健品和化妆品工业中。
微生物法合成L-茶氨酸具有环保、原料价格低廉、反应条件温和、产物易于分离的特点,逐渐受到研究人员的重视和广泛研究。如2015年12月30日公布的公布号为CN105200075A的“用于茶氨酸生产的质粒及其相应工程菌的构建与使用方法”专利,公开的方法是:在大肠杆菌中表达经过序列优化的目的基因谷氨酰甲胺合酶基因,以谷氨酸和乙胺为底物,微生物转化合成L-茶氨酸。用该方法合成L-茶氨酸的主要缺点是:(1)合成L-茶氨酸的最高浓度仅为12.2g/L,很难实现工业化。(2)底物需要谷氨酸和乙胺,增加了成本,同时乙胺对细胞和酶活性有抑制,导致L-产氨酸产量较低。(3)产物没有及时被转运蛋白转运出细胞,导致L-产氨酸产量较低。又如2019年02月22日公布的公布号为CN109370966A的“一种用于L-茶氨酸生产的基因工程菌及其发酵方法”专利,公开的方法是:在大肠杆菌基因组上单拷贝T7噬菌体的RNA聚合酶基因T7RNAP和双拷贝γ-谷氨酰甲胺合成酶基因gmas,敲除木糖操纵子阻遏蛋白基因xylR和琥珀酰CoA合成酶基因sucCD,以葡萄糖和乙胺为底物,发酵20h,L-茶氨酸产量为40g/L,糖酸转化率为25%。用该方法合成L-茶氨酸的主要缺点是:(1)发酵生产L-茶氨酸的最高浓度为40g/L,很难实现工业化生产。(2)发酵需要添加乙胺,乙胺对细胞和酶活性有抑制作用,导致L-产氨酸产量较低,同时添加乙胺导致成本增加。又如2019年05月21日公布的公布号为CN109777763A的“一株用于L-茶氨酸生产的基因工程菌及其构建与应用”专利,公开的方法是:在大肠杆菌基因组上单拷贝谷氨酸脱氢酶基因Cgl2079、丙酮酸羧化酶基因Cgl0689和柠檬酸合酶基因gltA以及三拷贝γ-谷氨酰甲胺合成酶基因gmas-Mu,工程菌株直接以葡萄糖为底物,发酵过程中流加乙胺溶液,发酵20h,5L发酵罐的L-茶氨酸产量为60g/L,糖酸转化率为40%。用该方法合成L-茶氨酸的主要缺点是:(1)发酵过程中需要流加乙胺,乙胺对细胞和酶活性有抑制作用,导致L-产氨酸产量较低,同时添加乙胺会导致成本增加。(2)γ-谷氨酰甲胺合成酶催化谷氨酸和乙胺合成L-茶氨酸是耗能过程,需要消耗当量的ATP,而该方法没有耦合ATP再生系统,导致L-茶氨酸产量不能进一步提高。
发明内容
本发明的目的是针对现有合成L-茶氨酸的菌株和方法的不足,提供一种基于多聚磷酸盐激酶PPK的ATP再生系统,同时过表达谷氨酰胺转运蛋白GNP1强化L-茶氨酸的转运效率,利用廉价的葡萄糖为底物,不需要额外添加乙胺,建立L-茶氨酸生物转化系统,提供一种用于生产L-茶氨酸的低成本,高效转化率的微生物发酵方法。
本发明的第一目的是提供一种用于生产L-茶氨酸的重组质粒,所述重组质粒为pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1,所述重组质粒包括Methyloversatilisuniversalis来源的γ-谷氨酰甲胺合成酶基因gmas、大肠杆菌来源的多聚磷酸盐激酶基因ppk、Bacillus subtilis来源的丙氨酸脱氢酶基因BsAld、Camellia sinensis来源的丙氨酸脱羧酶基因CsAlaDC和酿酒酵母来源的谷氨酰胺转运蛋白基因GNP1。
具体的,所述gmas基因的核苷酸序列如SEQ ID NO:1所示。
具体的,所述ppk基因的核苷酸序列如SEQ ID NO:2所示。
具体的,所述BsAld基因的核苷酸序列如SEQ ID NO:3所示。
具体的,所述CsAlaDC基因的核苷酸序列如SEQ ID NO:4所示。
具体的,所述GNP1基因的核苷酸序列如SEQ ID NO:5所示。
本发明的第二目的是在于提供的上述pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1重组质粒制备方法,具体技术方案如下:
上述pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1重组质粒的制备方法,具体步骤:
(1)设计引物:
SEQ ID NO:6,gmas-Ncol-F:5’-ATGAGCCCGAGCGAAGCCCAGC-3’
SEQ ID NO:7,gmas-BamHI-R:5’-AAAAAATTCCAGATAAGAATTC-3’
SEQ ID NO:8,ppk-SacI-F:5’-ATGGGTCAGGAAAAGCTATAC-3’
SEQ ID NO:9,ppk-HindIII-R:5’-TTCAGGTTGTTCGAGTGATTTG-3’
SEQ ID NO:10,BsAld-NdeI-F:5’-ATGATCATAGGGGTTCCTAAAG-3’
SEQ ID NO:11,BsAld-EcoRV-R:5’-AGCACCCGCAACAGATGACTC-3’
SEQ ID NO:12,CsAlaDC-EcoRV-F:5’-ATGGAAGGCACCGTGAGCGTTC-3’
SEQ ID NO:13,CsAlaDC-KpnI-R:5’-TTTGTGCAGGTCGCAATCAC-3’
SEQ ID NO:14,GNP1-KpnI-F:5’-ATGACGCTTGGTAATAGACG-3’
SEQ ID NO:15,GNP1-Xhol-R:5’-ACACCAGAAATCAAGAACTC-3’
(2)以合成的目的基因gmas、ppk、BsAld、CsAlaDC和GNP1为模板,进行PCR扩增。
(3)纯化PCR产物,连接到pETDuet-1载体上,得到所述pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1重组质粒。
本发明的第三目的是在于提供能产L-茶氨酸的含上述重组质粒的基因工程菌BL21(DE3)/pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1。
本发明的第四目的是在于利用上述方案中的基因工程菌制备L-茶氨酸的方法。
本发明的第五目的是在于提供了一种新型的γ-谷氨酰甲胺合成酶突变体,所述的突变体为:其氨基酸序列由SEQ ID NO:1突变而来,在选自下组的一个或两个氨基酸残基位点发生突变:174位和247位;其中,174位和247位突变可以选自其他19种氨基酸。
在具体的实施方式中,所述的γ-谷氨酰甲胺合成酶的氨基酸序列在选自下组的一个或两个位点突变为以下所示的氨基酸残基:
174位为丙氨酸、亮氨酸、脯氨酸、甘氨酸、赖氨酸。
247位为谷氨酸、丙氨酸、亮氨酸、脯氨酸、甘氨酸、赖氨酸。
本发明将Methyloversatilis universalis来源的γ-谷氨酰甲胺合成酶、大肠杆菌来源的多聚磷酸盐激酶PPK,Bacillus subtilis来源的丙氨酸脱氢酶BsAld,Camelliasinensis来源的丙氨酸脱羧酶CsAlaDC和酿酒酵母来源的谷氨酰胺转运蛋白GNP1克隆至质粒载体pETDuet-1上,获得重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1,再将重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1转入大肠杆菌BL21(DE3)中,获得用于生产L-茶氨酸的基因工程菌BL21(DE3)/pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1。
本发明的有益之处在于:本发明通过代谢工程手段,构建基于多聚磷酸盐激酶PPK的ATP再生系统,过表达谷氨酰胺转运蛋白GNP1强化L-茶氨酸的转运效率。同时γ-谷氨酰甲胺合成酶突变体,可以提高菌株L-茶氨酸的产量和转化率。本发明利用廉价的葡萄糖为底物,不需要额外添加乙胺,提供一种用于生产L-茶氨酸的低成本,高产量的微生物发酵方法,且在提高产量的同时,没有抑制菌株的生长,为大规模生产L-茶氨酸提供了新的策略。
具体实施方式
下面结合具体实施方式,进一步说明本发明。
实施例1
重组质粒为pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1及其制备方法。
本实施例提供一种用于生产L-茶氨酸的重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1,所述重组质粒包括Methyloversatilis universalis来源的γ-谷氨酰甲胺合成酶基因gmas、大肠杆菌来源的多聚磷酸盐激酶基因ppk、Bacillus subtilis来源的丙氨酸脱氢酶基因BsAld、Camellia sinensis来源的丙氨酸脱羧酶基因CsAlaDC和酿酒酵母来源的谷氨酰胺转运蛋白基因GNP1。
具体的,所述gmas基因的核苷酸序列如SEQ ID NO:1所示。
具体的,所述ppk基因的核苷酸序列如SEQ ID NO:2所示。
具体的,所述BsAld基因的核苷酸序列如SEQ ID NO:3所示。
具体的,所述CsAlaDC基因的核苷酸序列如SEQ ID NO:4所示。
具体的,所述GNP1基因的核苷酸序列如SEQ ID NO:5所示。
上述重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1的制备方法,具体技术方案如下,首先设计引物:
SEQ ID NO:6,gmas-Ncol-F:5’-ATGAGCCCGAGCGAAGCCCAGC-3’
SEQ ID NO:7,gmas-BamHI-R:5’-AAAAAATTCCAGATAAGAATTC-3’
SEQ ID NO:8,ppk-SacI-F:5’-ATGGGTCAGGAAAAGCTATAC-3’
SEQ ID NO:9,ppk-HindIII-R:5’-TTCAGGTTGTTCGAGTGATTTG-3’
SEQ ID NO:10,BsAld-NdeI-F:5’-ATGATCATAGGGGTTCCTAAAG-3’
SEQ ID NO:11,BsAld-EcoRV-R:5’-AGCACCCGCAACAGATGACTC-3’
SEQ ID NO:12,CsAlaDC-EcoRV-F:5’-ATGGAAGGCACCGTGAGCGTTC-3’
SEQ ID NO:13,CsAlaDC-KpnI-R:5’-TTTGTGCAGGTCGCAATCAC-3’
SEQ ID NO:14,GNP1-KpnI-F:5’-ATGACGCTTGGTAATAGACG-3’
SEQ ID NO:15,GNP1-Xhol-R:5’-ACACCAGAAATCAAGAACTC-3’
然后以基因合成技术合成的基因gmas、ppk、BsAld、CsAlaDC和GNP1为模板,进行PCR扩增。最后,纯化PCR产物,连接到pETDuet-1载体上,得到所述pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1重组质粒。
实施例2
构建γ-谷氨酰甲胺合成酶gmas基因第174位氨基酸密码子突变的突变体。
为了对γ-谷氨酰甲胺合成酶gmas基因的第174位氨基酸密码子进行野生型以外的19中突变,设计了引物E174A、E174R、E174N、E174D、E174C、E174Q、E174G、E174H、E174I、E174L、E174K、E174M、E174F、E174P、E174S、E174T、E174W、E174Y、E174V、E174-R。引物序列如下所示:
SEQ ID NO:16,E174A:5’-GCTGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:17,E174R:5’-CGAGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:18,E174N:5’-AACGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:19,E174D:5’-GACGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:20,E174C:5’-TGCGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:21,E174Q:5’-CAGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:22,E174G:5’-GGTGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:23,E174H:5’-CATGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:24,E174I:5’-ATCGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:25,E174L:5’-CTGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:26,E174K:5’-AAGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:27,E174M:5’-ATGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:28,E174F:5’-TTCGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:29,E174P:5’-CCTGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:30,E174S:5’-TCGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:31,E174T:5’-ACTGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:32,E174W:5’-TGGGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:33,E174Y:5’-TACGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:34,E174V:5’-GTTGTGGGCATCGATGTTTACCAG-3’
SEQ ID NO:35,E174-R:5’-ACGCAGAGATTCGCTCAGACG-3’
以重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1为模板,利用上述引物进行PCR扩增,然后纯化PCR产物,进行平端连接,然后进行转化、培养。将获得的单克隆进行测序确认,最终获得E174A、E174R、E174N、E174D、E174C、E174Q、E174G、E174H、E174I、E174L、E174K、E174M、E174F、E174P、E174S、E174T、E174W、E174Y和E174V共19中突变体。
实施例3
构建γ-谷氨酰甲胺合成酶gmas基因第247位氨基酸密码子突变的突变体。
为了对γ-谷氨酰甲胺合成酶gmas基因的第247位氨基酸密码子进行野生型以外的19中突变,设计了引物N247A、N247R、N247E、N247D、N247C、N247Q、N247G、N247H、N247I、N247L、N247K、N247M、N247F、N247P、N247S、N247T、N247W、N247Y、N247V、N247-R。引物序列如下所示:
SEQ ID NO:36,N247A:5’-GCTGATGGCAAACGTAACCTG-3’
SEQ ID NO:37,N247R:5’-CGAGATGGCAAACGTAACCTG-3’
SEQ ID NO:38,N247E:5’-GAGGATGGCAAACGTAACCTG-3’
SEQ ID NO:39,N247D:5’-GACGATGGCAAACGTAACCTG-3’
SEQ ID NO:40,N247C:5’-TGCGATGGCAAACGTAACCTG-3’
SEQ ID NO:41,N247Q:5’-CAGGATGGCAAACGTAACCTG-3’
SEQ ID NO:42,N247G:5’-GGTGATGGCAAACGTAACCTG-3’
SEQ ID NO:43,N247H:5’-CATGATGGCAAACGTAACCTG-3’
SEQ ID NO:44,N247I:5’-ATCGATGGCAAACGTAACCTG-3’
SEQ ID NO:45,N247L:5’-CTGGATGGCAAACGTAACCTG-3’
SEQ ID NO:46,N247K:5’-AAGGATGGCAAACGTAACCTG-3’
SEQ ID NO:47,N247M:5’-ATGGATGGCAAACGTAACCTG-3’
SEQ ID NO:48,N247F:5’-TTCGATGGCAAACGTAACCTG-3’
SEQ ID NO:49,N247P:5’-CCTGATGGCAAACGTAACCTG-3’
SEQ ID NO:50,N247S:5’-TCGGATGGCAAACGTAACCTGG-3’
SEQ ID NO:51,N247T:5’-ACTGATGGCAAACGTAACCTG-3’
SEQ ID NO:52,N247W:5’-TGGGATGGCAAACGTAACCTG-3’
SEQ ID NO:53,N247Y:5’-TACGATGGCAAACGTAACCTG-3’
SEQ ID NO:54,N247V:5’-GTTGATGGCAAACGTAACCTG-3’
SEQ ID NO:55,N247-R:5’-GATGCTCATGTGCATGTGCAG-3’
以重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1为模板,利用上述引物进行PCR扩增,然后纯化PCR产物,进行平端连接,然后进行转化、培养。将获得的单克隆进行测序确认,最终获得N247A、N247R、N247E、N247D、N247C、N247Q、N247G、N247H、N247I、N247L、N247K、N247M、N247F、N247P、N247S、N247T、N247W、N247Y和N247V共19中突变体。
实施例4
基因工程菌株发酵生产L-产氨酸。
从-80℃冰箱中取出菌株,用接种环挑一环重组大肠杆菌BL21(DE3)/pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1甘油菌,在含氨苄青霉素的LB固体培养基平板上划线,37℃恒温倒置培养12~16h。挑取单菌落接种子含有5mL LB液体培养基(含氨苄青霉素100μg/mL)的试管中,37℃、220RPM培养8~10h。随后将试管中菌种按照1%的接种量接种于200LB液体培养基(含氨苄青霉素100μg/mL,50g/L葡萄糖)中,37℃、220RPM培养8~10h。随后将菌种按照10%接种量接种于装2.8L LB液体培养基(含氨苄青霉素100μg/mL)的5L发酵罐中,发酵过程中控制pH稳定在7.0左右,温度维持在37℃,溶氧在25~35%之间。当OD600达到10时,加入终浓度1.0mM的IPTG,降温至30℃培养。当培养基中的葡萄糖消耗完之后,流加500g/L的葡萄糖溶液并维持发酵培养基中的葡萄糖浓度小于2g/L,发酵周期48h。发酵48h,L-茶氨酸产量为63.2g/L。
实施例5
γ-谷氨酰甲胺合成酶gmas基因第174位突变对L-茶氨酸合成的影响。
从-80℃冰箱中取出菌株,用接种环挑一环γ-谷氨酰甲胺合成酶gmas基因第174位突变的重组大肠杆菌,在含氨苄青霉素的LB固体培养基平板上划线,37℃恒温倒置培养12~16h。挑取单菌落接种于含有5mL LB液体培养基(含氨苄青霉素100μg/mL)的试管中,37℃、220RPM培养8~10h。随后将试管中菌种按照1%的接种量接种于200LB液体培养基(含氨苄青霉素100μg/mL,50g/L葡萄糖)中,37℃、220RPM培养8~10h。随后将菌种按照10%接种量接种于装2.8L LB液体培养基(含氨苄青霉素100μg/mL)的5L发酵罐中,发酵过程中控制pH稳定在7.0左右,温度维持在37℃,溶氧在25~35%之间。当OD600达到10时,加入终浓度1.0mM的IPTG,降温至30℃培养。当培养基中的葡萄糖消耗完之后,流加500g/L的葡萄糖溶液并维持发酵培养基中的葡萄糖浓度小于2g/L,发酵周期48h。发酵48h,L-茶氨酸产量为76.4g/L。
实施例6
γ-谷氨酰甲胺合成酶gmas基因第247位突变对L-茶氨酸合成的影响。
从-80℃冰箱中取出菌株,用接种环挑一环γ-谷氨酰甲胺合成酶gmas基因第247位突变的重组大肠杆菌,在含氨苄青霉素的LB固体培养基平板上划线,37℃恒温倒置培养12~16h。挑取单菌落接种于含有5mL LB液体培养基(含氨苄青霉素100μg/mL)的试管中,37℃、220RPM培养8~10h。随后将试管中菌种按照1%的接种量接种于200LB液体培养基(含氨苄青霉素100μg/mL,50g/L葡萄糖)中,37℃、220RPM培养8~10h。随后将菌种按照10%接种量接种于装2.8L LB液体培养基(含氨苄青霉素100μg/mL)的5L发酵罐中,发酵过程中控制pH稳定在7.0左右,温度维持在37℃,溶氧在25~35%之间。当OD600达到10时,加入终浓度1.0mM的IPTG,降温至30℃培养。当培养基中的葡萄糖消耗完之后,流加500g/L的葡萄糖溶液并维持发酵培养基中的葡萄糖浓度小于2g/L,发酵周期48h。发酵48h,L-茶氨酸产量为72.8g/L。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以权利要求为准。
Claims (4)
1.一种用于生产L-茶氨酸的重组质粒、基因工程菌株及方法,其特征在于:在大肠杆菌BL21(DE3)中过量表达Methyloversatilis universalis来源的γ-谷氨酰甲胺合成酶基因gmas、大肠杆菌来源的多聚磷酸盐激酶基因ppk、Bacillus subtilis来源的丙氨酸脱氢酶基因BsAld、Camellia sinensis来源的丙氨酸脱羧酶基因CsAlaDC和酿酒酵母来源的谷氨酰胺转运蛋白基因GNP1。
2.一种用于生产L-茶氨酸的基因工程菌构建方法,其特征在于:步骤如下:
(1)设计引物:
SEQ ID NO:6,gmas-Ncol-F:5’-ATGAGCCCGAGCGAAGCCCAGC-3’
SEQ ID NO:7,gmas-BamHI-R:5’-AAAAAATTCCAGATAAGAATTC-3’
SEQ ID NO:8,ppk-SacI-F:5’-ATGGGTCAGGAAAAGCTATAC-3’
SEQ ID NO:9,ppk-HindIII-R:5’-TTCAGGTTGTTCGAGTGATTTG-3’
SEQ ID NO:10,BsAld-NdeI-F:5’-ATGATCATAGGGGTTCCTAAAG-3’
SEQ ID NO:11,BsAld-EcoRV-R:5’-AGCACCCGCAACAGATGACTC-3’
SEQ ID NO:12,CsAlaDC-EcoRV-F:5’-ATGGAAGGCACCGTGAGCGTTC-3’
SEQ ID NO:13,CsAlaDC-KpnI-R:5’-TTTGTGCAGGTCGCAATCAC-3’
SEQ ID NO:14,GNP1-KpnI-F:5’-ATGACGCTTGGTAATAGACG-3’
SEQ ID NO:15,GNP1-Xhol-R:5’-ACACCAGAAATCAAGAACTC-3’
(2)以合成的目的基因gmas、ppk、BsAld、CsAlaDC和GNP1为模板,进行PCR扩增;
(3)纯化PCR产物,连接到pETDuet-1载体上,得到所述pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1重组质粒;
(4)将重组质粒pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1转入大肠杆菌BL21(DE3),得到所述产L-茶氨酸的基因工程菌BL21(DE3)/pETDuet-1-gmas-ppk-BsAld-CsAlaDC-GNP1。
3.一种新型的γ-谷氨酰甲胺合成酶突变体,其特征在于:其氨基酸序列由SEQ ID NO:1突变而来,在选自下组的一个或两个氨基酸残基位点发生突变:174位和247位;其中,174位和247位突变可以选自其他19种氨基酸。
4.权利要求1-2中所述的表达载体和基因工程菌或权利要求3所述的γ-谷氨酰甲胺合成酶在生产L-茶氨酸中的应用。
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