为了实现γ-氨基丁酸(GABA)在微生物中“一步法”高效生产,本研究构建出一株高产GABA的谷氨酸棒杆菌工程菌ATCC 13032/pDXW10-gadB1-gadB2,可以直接将自身合成的L-谷氨酸转变成GABA,并对该工程菌的发酵培养基和发酵条件进行了初步优化.结果表明:培养7 h～8h的种子液按发酵起始OD562=1.6转接至发酵培养基(葡萄糖、玉米浆分别100 g/L、4g/L),10 h添加PLP至0.1 mmol/L,发酵结束后胞外GABA可达(26.39±1.68) g/L.为工业化“一步法”生产GABA提供理论和实验基础.

Glutamate decarboxylase (GAD) transforms L-glutamate into gamma-aminobutyric acid (GABA) with the consumption of a proton. GAD derived from lactic acid bacteria exhibits optimum activity at pH 4.0-5.0 and significantly loses activity at near-neutral pH. To broaden the active range of the GAD GadB1 from Lactobacillus brevis Lb85 toward a near-neutral pH, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. For directed evolution of GadB1, a sensitive high-throughput screening strategy based on a pH indicator was established. One improved mutant, GadB1(T171/D294G/Q346H), was selected from 800 variants after one round of EP-PCR. It exhibited 3.9- and 25.0-fold increase in activity and catalytic efficiency, respectively at pH 6.0. Through site-specific mutagenesis, several improved mutants were obtained, with GadB1(E312S) being the best one. The combined mutant GadB1(T171/D294G/E312S/Q346H) showed even higher catalytic efficiency, 13.1- and 43.2-fold that of wild-type GadB1 at pH 4.6 and 6.0, respectively. The amount of GABA produced in gadB1(T171/D294G/Q346H_), gadB1(E312S_) and gadB1(T171/D294G/E312S/Q346H) expressing Corynebacterium glutamicum ATCC 13032 from endogenous L-glutamate increased by 9.6%, 20.3% and 63.9%, respectively. These results indicate that these mutations have beneficial effects on expanding the active pH range and on GABA biosynthesis, suggesting these GadB1 variants as potent candidates for GABA production. (C) 2014 Elsevier Inc. All rights reserved.

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes ( gadB1 and gadB2 ) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1 – gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of l -glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L −1 , and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L −1 after 84-h cultivation. Under optimal urea supplementation, l -glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L −1 after 120-h flask cultivation and 26.32 g L −1 after 60-h fed-batch fermentation. The conversion ratio of l -glutamate to GABA reached 0.60–0.74 mol mol −1 . By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated l -glutamate.