Metabolic Engineering Of Corynebacterium Glutamicum For γ-aminobutyric Acid And Its Precursor L-glutamate Biosynthesis

γ-Aminobutyric acid(GABA), which has a variety of physiological functions, is widely used in food, pharmaceutical fields and other industries. It is synthesized by glutamate decarboxylase(GAD), which catalyzes the α-decarboxylation of L-glutamate. Two GAD genes(gad B1 and gad B2) were co-expressed previously in an L-glutamate producing strain Corynebacterium glutamicum ATCC13032. The GABA production reached 18.7±2.1 g/L. To further enhance GABA production, metabolic engineering strategies are used here to improve the supply of the GABA precursor L-glutamate. The main research contents and results are as follows:(1) Firstly, the transcription levels of some genes related to L-glutamate biosynthesis were investigated by real-time PCR analysis in recombinant strain ATCC13032/p DXW-10-gad B1-gad B2 constructed previously. Three genes, i.e. odh A(encodes the E1 subunit of 2-oxoglutarate dehydrogenase), pyc(encodes pyruvate carboxylase) and pkn G(encodes serine/threonine-protein kinase) were determined to be deleted.(2) Three plasmids for deleting odh A, pyc and pkn G were constructed. Subsequently, these plasmids were transformed into C. glutamicum ATCC13032 separately and three deletion strains, i.e. Δodh A, Δpyc and Δpkn G were constructed. Next, the newly constructed plasmid p JYW-4-gad B1-gad B2 was transformed into three deletion mutants and the wild-type ATCC13032, generating four new recombinant C. glutamicum strains, i.e. odh A deleted GAD strain SNW201, pyc deleted GAD strain SNW202, pkn G deleted GAD strain SNW203 and wild type GAD strain SNW200.(3) The odh A deleted GAD strain SNW201 and the control strain SNW200 were fermented in fermenters. When odh A was deleted, the production of L-glutamate and GABA improved greatly. At 72 h of fermentation, GABA production of SNW201 increased to 29.5±1.1 g/L, 51.8% higher than that of SNW200. The total amount of L-glutamate and GABA reached 0.29 M, 31.8% higher than that of SNW200.(4) The pyc deleted GAD strain SNW202 was fermented in a fermenter. When pyc gene was deleted, the production of L-glutamate and GABA improved a little as compared to wild type strain SNW200. At 72 h of fermentation, GABA production increased to 24.9±0.7 g/L, 28.3% higher than that of SNW200. The total amount of L-glutamate and GABA reached 0.25 M, 13.6% higher than that of SNW200.(5) Finally, the pkn G deleted GAD strain SNW203 was fermented. When pkn G gene was deleted, the production of L-glutamate and GABA improved significantly as compared to strain SNW200. At 72 h of fermentation, GABA production increased to 30.2±0.3 g/L, 55.4% higher than that of SNW200. The total amount of L-glutamate and GABA reached 0.30 M, 36.4% higher than that of SNW200.The production have improved because of the deletion of the three genes.