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...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2021-05, Vol.105 (10), p.4127-4140
Main Authors: Lyu, Changjiang, Yao, Lili, Zhu, Qi, Mei, Jiaqi, Cao, Yucheng, Hu, Sheng, Zhao, Weirui, Huang, Jun, Mei, Lehe, Yao, Shanjing, Du, Guocheng
Format: Article
Language:English
Subjects:
Bacteria
Bioactive compounds
Biomedical and Life Sciences
Biosynthesis
Biotechnological Products and Process Engineering
Biotechnology
Catalysts
Catalytic activity
Cell growth
Decarboxylation
E coli
Escherichia coli
Fermentation
Food
Glutamate decarboxylase
Glutamic acid
Homology
Lactic acid
Lactic acid bacteria
Lactobacillus plantarum
Lactococcus lactis
Life Sciences
Microbial Genetics and Genomics
Microbiology
Microorganisms
Mutants
pH control
pH effects
γ-Aminobutyric acid
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Summary: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 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 μM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadB ΔC10 , GadB ΔC11 , GadB ΔC12 , GadB ΔC13 ) were constructed based on homology modeling. Among them, the mutant GadB ΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadB ΔC11 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 ΔC11 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. 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.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-021-11328-5