Enhanced fermentative γ-aminobutyric acid production by a metabolic engineered Corynebacterium glutamicum

Abstract

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with important physiological functions, which has been widely used in food, pharmaceuticals, and polyamides production. The fermentative GABA production by Corynebacterium glutamicum was recognized as one of the most promising methods. However, the problems of low catalytic activity of the heterologously expressed glutamate decarboxylase (GAD) and the imbalanced carbon flux between cell growth and GABA synthesis severely limited the GABA production by C. glutamicum. This study applied combinational metabolic engineering and catalytic condition optimization strategies to solve these two major obstacles. The secretory expression of GAD was enhanced using a bicistronic-designed expression cassette. This bicistronic expression cassette was further triply inserted into the genome by substituting the ldhA, pqo, and ack genes, thus stabilizing the expression of GAD and reducing the accumulation of by-products of lactate and acetate. A growth-regulated promoter P_{CP_2836} was applied to dynamically control the expression of odhA, thus controlling the α-oxoglutarate dehydrogenase complex activity for balanced cell growth and GABA production. The glutamate precursor synthesis and pyridoxal 5′-phosphate supply were also strengthened by promoter substitution. Finally, through a two-stage pH-controlled fed-batch fermentation under optimized conditions, the engineered strain reached GABA titer of 81.31 ± 1.31 g/L with a yield and productivity of 0.50 ± 0.01 g/g and 1.36 ± 0.23 g L⁻¹ h⁻¹, which was 4.8%, 13.6%, and 11.2% higher than that of the original strain. This study laid a solid foundation for industrial fermentative GABA production by engineered C. glutamicum.