Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli

Abstract

BackgroundGamma (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 Escherichia coli and lactic acid bacteria. However, no GADs from Streptomyces have been characterized. The present study is aimed to identify new GADs from Streptomyces strains and establish an efficient bioproduction platform for GABA in E. coli using these enzymes.ResultsBy sequencing and analyzing the genomes of three Streptomyces strains, three putative GADs were discovered, including StGAD from Streptomyces toxytricini NRRL 15443, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982. The corresponding genes were cloned from these strains and heterologously expressed in E. coli BL21(DE3). The purified GAD proteins showed a similar molecular mass to GadB from E. coli 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 Vmax, Km, kcat and kcat/Km values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with kcat/Km values of 0.62 and 1.21 mM− 1·s− 1, respectively. In addition, engineered E. coli strains harboring StGAD, SsGAD and ScGAD were used as whole-cell biocatalysts for production of GABA from L-Glu. E. coli/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.ConclusionsThree new GADs have been functionally characterized from Streptomyces, among which two showed higher catalytic efficiency than previously reported GADs. Engineered E. coli harboring SsGAD provides a promising cost-effective bioconversion system for industrial production of GABA.Electronic supplementary materialThe online version of this article (10.1186/s13036-019-0154-7) contains supplementary material, which is available to authorized users.

Efficient production of gamma‐aminobutyric acid by engineered Saccharomyces cerevisiae with glutamate decarboxylases from Streptomyces

“…Proton requirement for GAD‐catalyzed glutamate decarboxylation and its role in maintaining the cellular pH environment make pH an important factor for the GAD activity and thus GABA production . While the optimal pH for purified SsGAD was found to be 3.8 in our previous study , the optimal pH for GABA production in S. cerevisiae BJ5464/SsGAD was 5.4 (Fig. 3A).…”

“…PLP is well known as an indispensable cofactor accelerating GAD‐catalyzed decarboxylation of l ‐Glu via binding on the specific site in GADs . Thus, we investigated the influence of PLP concentrations (0–1 mM) on the conversion of l ‐Glu to GABA.…”

“…The supernatants were analyzed through SDS‐PAGE with 12% separation gel and 4% stacking gel. Laemmli's Tris–glycine electrolyte buffer at pH 8.3 was used as running buffer . The BLUEstain TM protein marker was purchased from Gold Biotechnology (St. Louis, MO, USA).…”

“…GABA is also widely used as food and feed supplements . Naturally, GABA is synthesized from l ‐glutamate ( l ‐Glu) through enzymatic decarboxylation by glutamate decarboxylase (GAD; EC4.1.1.15) . GAD is a pyridoxal 5′‐phosphate (PLP)‐dependent decarboxylase widespread in bacteria, yeast, and filamentous fungi .…”

Microbial Production and Enzymatic Biosynthesis of γ-aminobutyric acid (GABA) Using<em> Lactobacillus plantarum</em> FNCC 260 Isolated From Indonesian Fermented Foods

“…The GAD assay was carried out using colorimetric method [37]. The reaction mixtures consist of 200 mM Na2HPO4-citric acid buffer (pH 5.0), 20 mM L-MSG, 0.2 mM PLP and 20 µl of purified GAD.…”