Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40 degrees C and pH 5.0. The K-m value and catalytic efficiency (k(cat)/K-m) of LpGadB for L-Glu was 21.33 mM and 1.19 mM(-1)s(-1), respectively, with the specific activity of 26.67 mu M/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadB(Delta C10), GadB(Delta C11), GadB(Delta C12), GadB(Delta C13)) were constructed based on homology modeling. Among them, the mutant GadB(Delta C11) with highest catalytic activity at near-neutral pH values was selected. In further, the GadB(Delta C11) and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadB(Delta C11)C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.

Automated summary

In this study, the bioactive compound GABA was successfully biosynthesized using lactic acid bacteria as catalysts, overcoming the discrepancy in optimal pH for GAD activity and cell growth to achieve high GABA production. Enhanced GAD activity was achieved through protein engineering strategies, and a two-stage pH control strategy was employed to increase GABA yield, resulting in the highest yield obtained to date for GABA fermentation with Lactococcus lactis as a microbial cell factory.