Enhanced Production Of γ-aminobutyrate In Lactococcus Lactis By Over-expressing GAD System Key Genes

Gmama-aminobutyric acid (GABA) is a natural non-protein amino acid, which is widely distributed in plants, animals and microorganisms. As the major inhibitory neurotransmitter in the central nervous system, GABA has many physiological functions, including induction of hypotension, tranquilizing excitement and regulating hormone secretion. Among these attempts, biocatalytic synthesis of GABA is a more promising way due to its high catalytic efficiency and environmental compatibility. Escherchia coli, with its advantages of rapid growth and high production of GABA, becomes a popular strain, but is not applied in food industry owing to potential security risk. Considering the safety for food and health, lactic acid bacteria (LAB) rather than E. coli, are preferred for GABA preparation.Lactococcus lactis GAD system consists of a glutamate decarboxylase (GAD) and a Glu-GABA antiporter, the coding genes are named gadB and gadC, respectively. GAD catalyzes the irreversible reaction of L-glutamate to GABA, while Glu-GABA antiporter transfers L-glutamate into the cell and delivers GABA out of the cell. In the dissertation, the model strain Lactococcus lactis NZ9000 in cell factories was used as host cells, the key gene of GAD system from Lc. lactis subsp. lactis CV56 was overexpressed for GABA biosyhthesis by metabolic engineering, and microbial physiological and biochemical analysis. The reaction conditions for GABA biosynthesis were investigated, and biosynthesis of GABA using free cells was studied in the optimized conditions.Firstly, nucleotide sequencing analysis of Lc. lactis subsp. lactis CV56 showed that the gadB gene comprised comprised 1401 bp and encoded a 466-amino acid protein with molecular weight of 53.8 kDa. The protein sequence showed high identity with GADs from other LABs. GADcvs6 has the conserved lysine residue (K277), threonine residues (T213) and aspartic acid residue (D245), as well as a highly conserved motif H(I/V)DAASGG. Then the gadB gene was over-expressed in E.coli BL21 (DE3) by using pET-28a (+), and the purified recombinant GAD was acquired through Ni-NTA affinity chromatography. The optimum pH and temperature for the recombinant enzyme were 4.7 and 50℃. The Km and Vmax were 3.88 mM and 58.66 U/mg, respectively.Secondly, the gadB and gadCB genes of Lc. lactis subsp. lactis CV56 were cloned into Lc. lactis NZ9000, respectively, and the recombinant strains were named Lc. lactis NZ9000:pNZ8148-gadB and Lc. lactis NZ9000:pNZ8148-gadCB. Using Lc. lactis NZ9000:pNZ8148-gadCB strain for fermentation, the GABA yield was 7.06-fold than Lc. lactis NZ9000 after 45h induction; using Lc. lactis NZ9000:pNZ8148-gadCB strain for fermentation, the GABA yield of fermentation broth was 16.63-fold than the Lc. lactis NZ9000 strain at the same fermentation condition. By cloning gadB gene of Lc. lactis subsp. lactis CV56 into the expression vector pNZ8149, a food-grade recombinant strain Lc. lactis NZ3900:pNZ8149-gadB was constructed to apply for food industry.Thirdly, the influence of cell age, buffer system, temperature, pH and PLP concentration on GABA biosynthesis were investigated in Lc. lactis NZ9000-pNZ8148-gadCB. The optimum reaction system was HAc-NaAc buffer (pH 4.2), temperature 45 ℃, without addition of PLP, and the bacteria cells was harvested after inducing for 4 h. After 60 h reaction at the optimum conditions, using Lc. lactis NZ9000:pNZ8148-gadB and Lc. lactis NZ9000:pNZ8148-gadCB, the GABA yield reached 37.71 g/L and 43.32 g/L, which were 3.97-fold and 4.56-fold than control, respectively.