Abstract
Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L−1 glucose and 0.1 mM pyridoxal 5′-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L−1 (0.259 g L−1 h−1) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol−1.
Highlights
Diverse microorganisms, animals, and plants synthesize the amino acid gamma-aminobutyric acid (GABA), which does not naturally occur in proteins
Effect of exogenous L-glutamate on GABA fermentation by C. glutamicum Glutamate decarboxylase (GAD) To examine the effect of glutamate as a precursor for GABA synthesis, L-glutamate was added to a culture of C. glutamicum GAD grown on GABA Production 2 (GP2) medium with glucose as the primary carbon source, soy peptone as the nitrogen source, and the GAD cofactor pyridoxal 5′-phosphate (PLP) (Figure 1)
Influence of the pknG deletion on GABA synthesis We examined the effect of the pknG deletion on GABA production from glucose, using C. glutamicum strains GAD, GADΔpknG, and W
Summary
Animals, and plants synthesize the amino acid gamma-aminobutyric acid (GABA), which does not naturally occur in proteins. GABA functions as a neurotransmitter in humans, lowers blood pressure (Hayakawa et al 2004), and is a component of pharmaceuticals and foods (Li and Cao 2010). The bioplastic polyamide 4 (PA4) is a linear polymer of GABA, which is chemically synthesized from the GABA lactam 2pyrrolidone (Kawasaki et al 2005). The synthesis of GABA from abundantly available biomass by recombinant microorganisms will make it possible to produce new bioplastics at low cost. Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the conversion of L-glutamate to GABA through alphadecarboxylation (Fonda 1985). Lactic acid bacteria produce GABA when glutamate is added to the fermentation medium. The quantities of GABA produced by this method are sufficient for producing foods, it is not cost-effective for producing chemicals
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