Abstract: In this study, we addressed the effects of exogenous addition of gamma-aminobutyric acid (GABA) and overexpression of glutamic acid decarboxylase (CsGAD) gene from Camellia sinensis on acid and cold stress tolerance in Lactobacillus lactis NZ9000. The results showed that exogenous addition of GABA had no significant beneficial effect on the biomass of L. lactis NZ9000 under both acid and cold stress. The CsGAD gene was obtained by reversed transcription PCR (RT-PCR) and cloned into the expression vector pNZ8148-CsGAD for efficient expression of GAD in L. lactis NZ9000/pNZ8148-CsGAD. The GABA synthesis ability of the recombinant strain was increased by over 5 times after 8 h of induction with nisin as determined by HPLC. Growth curves showed that under normal culture conditions, the recombinant strain reached the logarithmic phase of growth later than the control strain and the?maximum?yield of?biomass?at?the?stationary phase was 1.5 folds higher than that of the control strain. The control did not grow substantially at pH 4.0, while the recombinant strain remained viable. Under cold stress, the anti-freeze and survival ability of bacterial cells were higher in the late stationary growth phase than in the middle logarithmic phase and the early stationary phase. Moreover, the survival rate of the recombinant strain was 1 to 2 orders of magnitude higher than that of the control at each phase of growth. Taken collectively, the recombinant strain L. lactis NZ9000/pNZ8148-CsGAD had improved cold and acid stress tolerance due to GABA production from heterologous expression of CsGAD in it. The results of this study provide a basis for exploring the stress tolerance mechanism and improving the growth status of lactic acid bacteria for expanded industrial application.

Key words: Lactobacillus lactis, glutamic acid decarboxylase, gamma-aminobutyric acid, acidic stress, frozen stress