Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma‐aminobutyric acid

Abstract

Corynebacterium glutamicum has been used as a sustainable microbial producer for various bioproducts using cheap biomass resources. In this study, a high GABA-producing C. glutamicum strain was constructed by chromosomal editing. Lactobacillus brevis-derived gadB2 was introduced into the chromosome of C. glutamicum ATCC 13032 to produce gamma-aminobutyric acid and simultaneously blocked the biosynthesis of lactate and acetate. GABA transport and degradation in C. glutamicum were also blocked to improve GABA production. As precursor of GABA, l-glutamic acid synthesis in C. glutamicum was enhanced by introducing E. coli gdhA encoding glutamic dehydrogenase, and the copy numbers of gdhA and gadB2 were also optimized for higher GABA production. The final C. glutamicum strain CGY705 could produce 33.17 g/L GABA from glucose in a 2.4-L bioreactor after 78 h fed-batch fermentation. Since all deletion and expression of genes were performed using chromosomal editing, fermentation of the GABA-producing strains constructed in this study does not need supplementation of any antibiotics and inducers. The strategy used in this study has potential value in the development of GABA-producing bacteria.

Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli

“…In comparison with the de novo biosynthesis of GABA from glucose or glycerol without the addition of l -Glu or MSG, − the GABA produced from whole-cell bioconversion was much more effective. To the best of our knowledge, the GABA concentration of 70.6 g/L (with 1.01 g/L/h productivity) is the highest titer achieved thus far using the engineered C.…”

Model-Guided Metabolic Rewiring for Gamma-Aminobutyric Acid and Butyrolactam Biosynthesis in Corynebacterium glutamicum ATCC13032

“…In contrast, the removal of the above limitations for GABA synthesis via the GAD-based route had no significant effects on GABA titer as expected, indicating that GAD activity is still a key limiting factor for GABA production. Even if the pH threshold of GAD was broadened to 6.0 by deleting the C-terminal [ 28 , 29 ], the glutamate and GABA would not be synthesized simultaneously, due to the occurrence of glutamate biosynthesis at pH 6.7–7.5 [ 33 ]. Therefore, directed evolution of GAD to improve its enzymatic activity at neutral pH or two-stage fermentation based on the pH control will contribute to improve GABA production from glucose via the GAD-based biosynthetic route.…”

“…The optimization of plasmid-based GAD expression by a strong promoter and an optimal ribosomal binding site as well as co-expressing gadB1 and gadB2 genes efficiently improved GABA production [ 27 , 30 , 31 , 32 ]. Chromosomal integration of multiple copies of gdhA and gadB2 genes enhanced GABA production [ 33 ]. Another biological route for GABA biosynthesis from putrescine was constructed following a two-step enzymatic reaction catalyzed by patA -encoding putrescine aminotransferase (EC 2.6.1.82) and patD -encoding γ-aminobutyraldehyde dehydrogenase (EC 1.2.1.19) [ 34 ].…”