Abstract
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°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM−1s−1, respectively, with the specific activity of 26.67 μM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 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-gadBΔC11C 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.
Key points
• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.
• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.
• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.
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Funding
This work was supported by the National Natural Science Foundation of China (Nos. 31670804, 31971372), China Postdoctoral Science Foundation (2020M671337), Postdoctoral Foundation of Jiangsu Province (2020Z074), Ningbo “Scientific and Technological Innovation 2025” Key Project (2018B10093, 2020Z080), and the foundation of State Key Laboratory of Microbial Metabolism (MMLKF19-10).
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LJ designed all the experiments, analyzed the data, and wrote the manuscript. LJ, YL, HS, HJ, ZR, and ZQ generated the genetic constructs, conducted experiments, and analyzed the data. MH and YJ contributed with scientific discussions and commented on the manuscript. ML and DC supervised the work and revised the manuscript. All authors read and approved the final manuscript.
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Lyu, C., Yao, L., Zhu, Q. et al. Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid. Appl Microbiol Biotechnol 105, 4127–4140 (2021). https://doi.org/10.1007/s00253-021-11328-5
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DOI: https://doi.org/10.1007/s00253-021-11328-5