Identification of new glutamate decarboxylases from for efficient production of γ-aminobutyric acid in engineered .

Background: Gamma (γ)-Aminobutyric acid (GABA) as a bioactive compound is used extensively in functional foods, pharmaceuticals and agro-industry. It can be biosynthesized via decarboxylation of monosodium glutamate (MSG) or L-glutamic acid (L-Glu) by glutamate decarboxylase (GAD; EC4.1.1.15). GADs have been identified from a variety of microbial sources, such as and lactic acid bacteria. However, no GADs from have been characterized. The present study is aimed to identify new GADs from strains and establish an efficient bioproduction platform for GABA in using these enzymes.

Results: By sequencing and analyzing the genomes of three strains, three putative GADs were discovered, including StGAD from NRRL 15443, SsGAD from sp MJ654-NF4 and ScGAD from ATCC 49982. The corresponding genes were cloned from these strains and heterologously expressed in BL21(DE3). The purified GAD proteins showed a similar molecular mass to GadB from BL21(DE3). The optimal reaction temperature is 37 °C for all three enzymes, while the optimum pH values for StGAD, SsGAD and ScGAD are 5.2, 3.8 and 4.2, respectively. The kinetic parameters including , K, and /K values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with /K values of 0.62 and 1.21 mM·s, respectively. In addition, engineered strains harboring StGAD, SsGAD and ScGAD were used as whole-cell biocatalysts for production of GABA from L-Glu. /SsGAD showed the highest capability of GABA production. The cells were repeatedly used for 10 times, with an accumulated yield of 2.771 kg/L and an average molar conversion rate of 67% within 20 h.

Conclusions: Three new GADs have been functionally characterized from , among which two showed higher catalytic efficiency than previously reported GADs. Engineered harboring SsGAD provides a promising cost-effective bioconversion system for industrial production of GABA.