短乳杆菌NCL912 γ-氨基丁酸代谢支路解析及与三羧酸循环的偶联修饰

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammals, with a variety of important biological functions, and has been approved as a new resource food. The synthesis of GABA by food grade microorganisms is a new green technology. The GABA production of Lactobacillus brevis NCL912, an our previously isolated strain, reaches 104g/L with 48 h of fermentation (Li, et al. Microb Cell Fact. 2010, 9: 85), showing a good application prospect. However, our previous studies found that NCL912 also has the ability of degrading GABA, resulting in decrease in concentration of GABA. We speculated that GABA shunt exists in NCL912 (a collateral of tricarboxylic acid cycle), which converts GABA into succinate. This project aims to identify the genetic basis of GABA shunt in NCL912; elucidate the inherent correlation of the shunt and tricarboxylic acid cycle; construct a high-GABA yielding genetically engineered strain by knocking out the GABA shunt and strengthening the tricarboxylic acid cycle; research fermentation characteristics of the strain, optimize its culture conditions, and further enhance the strain’s ability of GABA accumulation through fermentation regulation. Our goals are to accumulate data for further strain modification, metabolic regulation and clarification of GABA’s physiological functions; and offer the reference to reveal GABA shunts of the other lactic acid bacteria.

γ-氨基丁酸（GABA）是哺乳动物体内主要的抑制性神经递质，具有多种重要的生物学功能，已被批准为新资源食品。利用食品级微生物合成GABA是一种新型绿色技术。短乳杆菌NCL912发酵48h GABA达104g/L（Li, et al. Microb Cell Fact. 2010, 9: 85），具有应用前景。然而，前期工作发现NCL912同时具有GABA降解能力，导致产物浓度降低。推测其中存在GABA代谢支路（三羧酸循环的侧支），将GABA转化为琥珀酸。本项目拟解析NCL912 GABA代谢支路的遗传基础；阐明该支路与三羧酸循环的关联；敲除GABA代谢支路，并强化TCA循环，构建高产GABA基因工程菌；研究菌种的发酵特性，优化培养条件，并通过发酵调控进一步提升产物积累能力。旨在为进一步改造菌种、代谢调控以及阐明GABA的生理功能积累数据；为揭示其它乳酸菌GABA代谢支路提供思路借鉴。

γ-氨基丁酸（Gamma-aminobutyric acid, GABA）是哺乳动物体内主要的抑制性神经递质，具有多种重要的生物学功能，已被批准为新资源食品。利用食品级微生物合成GABA是一种新型绿色技术。短乳杆菌NCL912发酵48h GABA达104g/L，具有应用前景。然而，前期工作发现NCL912同时具有GABA降解能力，导致产物浓度降低，推测NCL912中存在 GABA的降解途径。主要研究结果如下。.（1）明确了GABA代谢途径中的gadA和gadC基因，分别负责底物脱羧和产物运输。（2）提出了底物缓释自调控pH以提高GABA合成水平的策略，底物谷氨酸可以在发酵前一次性加入，一方面解除了底物抑制，另一方面在整个发酵过程中不需要补充酸液，发酵时间48 h后产物达205 g/L，比传统补料工艺提升了102%。（3）建立了高效、操作简单的基因组步移方法——引物阶梯式部分重叠PCR（Stepwise partially overlapping primer-based PCR，SWPOP-PCR），用于获取已知序列的侧翼。（4）建立了醋酸锌辅助差异沉淀/溶解法（zinc acetate-assisted differential precipitation/dissolution，ZA-DPD）分离底物谷氨酸，方法的关键是在每次沉淀过程中，谷氨酸沉淀试剂保证大部分GABA仍可溶，仅少量GABA与谷氨酸共沉淀；在耦合的溶解过程中，共沉淀的GABA完全溶出，同时仅微量或无谷氨酸溶出，这一过程重复两次，直到完全去除谷氨酸盐。ZA-DPD偶联分光光度法后，可对GABA进行定量。（5）还建立了无Cu2+洗脱预染纸色谱-分光光度法，用于GABA的高通量定量。.研究结果为菌种改造、代谢调控以及阐明GABA的生理功能奠定了基础，可能有助于整体提高乳酸菌的GABA合成水平。