Expression Of Lactobacillus Brevis Glutamate Decarboxylase And Synthesis Of γ-aminobutyric Acid In Corynebacterium Glutamicum

Glutamate decarboxylase (GAD) catalyses the irreversibleα-decarboxylation of L-glutamate to formγ-aminobutyric acid (GABA). GABA is an amino acid that is not incorporated into proteins and functions as a major inhibitory neurotransmitter in animals. It has many physiological properties related to anti-anxiety, hypotension, ataraxy, brain activity and so on. Therefore, GABA may function as a bioactive component in the food, feed, and pharmaceutical fields. In this work, the main contents and results are as follows.Firstly, a lactic acid bacterium stain, Lb85, capable of producing GABA was screened from the preserved lactic acid bacteria in our laboratory. This strain could produce 2.52 g/L GABA from 10 g/L L-glutamate precursor. It was a gram-positive, rod-typebacterium. After 16S rDNA sequencing, it was identified as Lactobacillus brevis. The L. brevis Lb85 was deposited into the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2010367.Secondly, two GAD genes, gadB1 and gadB2, as well as the L-glutamate/GABA antiporter gene gadC and regulator gene gadR in the upstream of gadB2 were amplified from L. brevis Lb85 and cloned into three shuttle expression vectors between Escherichia coli and C. glutamicum, that were, inducible vector pDXW-8, constitutive vector pDXW-9 with tac promoter and constitutive vector pDXW-10 with much strong tac-M promoter. Thus six recombinant plasmids, pDXW-8/gadB1, pDXW-8/gadB2, pDXW-8/gadCB2, pDXW-8/gadRCB2, pDXW-9/gadRCB2, pDXW-10/gadRCB2 were constructed. These plasmids were transformed into electrocompetent Corynebacterium glutamicum ATCC 13032, yielding the recombinant C. glutamicum strains ATCC 13032/pDXW-8/gadB1, ATCC 13032/pDXW-8/gadB2, ATCC 13032/pDXW-8/gadCB2, ATCC 13032/pDXW-8/gadRCB2, ATCC 13032/pDXW-9/gadRCB2 and ATCC13032/pDXW-10/gadRCB2.Thirdly, the producing ability of L-glutamate and GABA in ATCC 13032/pDXW-8/gadB2, ATCC 13032/pDXW-8/gadCB2 and ATCC 13032/pDXW-8/gadRCB2 under different glucose concentration in the flask fermentation were checked. The recombinant strain ATCC13032/pDXW-8/gadRCB2 showed the highest GABA production. However, the transcriptional level of gadB2 among these three strains and that of gadC between ATCC 13032/pDXW-8/gadCB2 and ATCC 13032/pDXW-8/gadRCB2 were nearly same, indicating that although the co-expression of gadR, gadC with gadB2 in C. glutamicum could not increase gadB2 and gadC transcription, it increased GABA accumulation and transport. The GABA production of all the six recombinant strains under the same culture condition at flask fermentation showed that GABA production of ATCC 13032/pDXW-10/gadRCB2 was the highest, indicating that the stronger promoter would be better for GAD expression and GABA production.Finally, optimization of culture conditions during flask fermentation was carried out using the recombinant C. glutamicum strains ATCC 13032/pDXW-10/gadRCB2. The final GABA production was 2.41 g/L after optimization of condition. This study improved the synthetic ability of C. glutamicum to produce functional products other than normal amino acids, that was GABA, and would be helpful for the further study.