Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase.

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).

Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli.

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.

Development of novel cell surface display in Corynebacterium glutamicum using porin.

We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.