Efficient one-step preparation of γ-aminobutyric acid from glucose without an exogenous cofactor by the designed Corynebacterium glutamicum

Lactobacillus plantarum CCTCC M209102 efficiently produces gamma-aminobutyric acid (GABA) from L-glutamate, in which glutamate decarboxylase and pyridoxal kinase are involved in the transformation. Pyridoxal kinase catalyzes ATP-dependent phosphorylation of pyridoxal to produce pyridoxal-5'-phosphate, which is the cofactor required for glutamate decarboxylase to biotransform GABA from L-glutamate. Corynebacterium glutamicum G01 is a good producer of L-glutamate from glucose. However, it cannot yield GABA from L-glutamate due to the absence of glutamate decarboxylase and pyridoxal kinase. In this work, to realize the efficient one-step preparation of GABA from glucose without exogenous pyridoxal-5'-phosphate, the metabolic module from L-glutamate to GABA based on glutamate decarboxylase and pyridoxal kinase in L. plantarum was grafted into C. glutamicum. To further improve the GABA production, the pathways to by-product pools of L-arginine, L-proline and L-lysine were blocked using the insertional mutation technique. The engineered C. glutamicum APLGGP carrying argB::tacgad, proB::tacgad and dapA::tacplk could efficiently convert glucose into GABA in one-step without an exogenous co-factor. In fed-batch cultures, the recombinant C. glutamicum APLGGP produced 70.6 g L-1 GABA at 30 degrees C and 70 h through a two-stage pH control strategy. To our knowledge, this is the highest reported GABA production using glucose as a substrate, and this designed C. glutamicum should be an excellent candidate for producing GABA on an industrial scale. This work is expected to pave the way to redesign the bioreactor for efficient one-step biosynthesis of GABA from glucose without an exogenous co-factor.