以葡萄糖为底物一步法合成γ-氨基丁酸整合型重组钝齿棒杆菌的构建

One-step fermentation converting glucose in γ-aminobutyric acid by a recombinant Corynebacterium crenatum

【目的】为了构建一株直接利用廉价的葡萄糖合成γ-氨基丁酸的重组钝齿棒杆菌,将来自于植物乳杆菌γ-氨基丁酸合成途径的关键酶谷氨酸脱羧酶基因(lpgad)在产谷氨酸菌株钝齿棒杆菌中进行整合表达,实现葡萄糖到GABA的一步法生产。【方法】运用PCR技术扩增得到带有tac启动子的谷氨酸脱羧酶基因tacgad。通过重叠PCR的方法获得钝齿棒杆菌精氨酸合成途径关键酶N-乙酰谷氨酸激酶(NAGK)基因内部缺失型基因ΔargB。利用自杀载体pK18mobsacB构建同源整合载体pK18-ΔargB::tacgad,以ΔargB的上下游序列为同源臂,通过两次同源重组将tacgad基因整合到钝齿棒杆菌基因组,同时将NAGK基因argB灭活,利用蔗糖致死基因sacB反向筛选标记筛选得到谷氨酸脱羧酶的重组钝齿棒杆菌C.crenatumΔargB::tacgad。重组钝齿棒杆菌以葡萄糖为底物进行发酵,测定GABA含量。【结果】重组菌C.crenatumΔargB::tacgad成功表达谷氨酸脱羧酶,同时阻断了精氨酸合成途径对谷氨酸到GABA代谢途径的竞争,粗酶液基本检测不到NAGK活性,发酵液无精氨酸合成。通过96 h发酵,重组菌可积累约8.28 g/L的GABA。【结论】本研究通过将谷氨酸脱羧酶基因定向整合到钝齿棒杆菌精氨酸合成途径的关键酶基因argB内部,成功表达谷氨酸脱羧酶的同时阻断竞争途径精氨酸的合成。本研究为实现直接利用葡萄糖合成GABA的一步法生产奠定了基础。

[ Objective] We constructed an integrative recombinant Corynebacterium crenatum that could directly convert glucose in γ-aminohutyric acid （GABA）. [ Methods ] Using overlap PCR, we obtained AargB fragment that lacked 491 bp of N-acetylglutamate kinase （NAGK） gene. The glutamate decarboxylase （GAD） gene attached tac promoter was amplified and inserted into AargB fragment. Using the upstream and downstream of AargB as homologous arms, we constructed pK18-△argB： ： tacgad which was used for the integration of tacgad gene onto the C. crenatum genome. Through two homologous recombinations, we got an argB blocked strain C. crenatum △argB：： tacgad which could successfully express GAD. We also fermented the recombinant strain with glucose as substrate and the production of GABA was detected. [ Results] In C. crenatum △argB：：tacgad, NAGK was totally inactivated and no L-arginine was detected though L-glutamic acid was accumulated. As a result of the efficient expression of GAD, part of L-glutamic acid was transformed into GABA, and the final yield of GABA was about 8.28 g/L. [ Conclusion] We successfully constructed a recombinant strain that could efficiently produce GABA by one-step fermentation from glucose. This research provided a new approach for GABA production.