Whole-cell conversion of l-glutamic acid into gamma-aminobutyric acid by metabolically engineered Escherichia coli

Abstract

A simple and high efficient way for the synthesis of gamma-aminobutyric acid (GABA) was developed by using engineered Escherichia coli as whole-cell biocatalyst from l-glutamic acid (l-Glu). Codon optimization of Lactococcus lactis GadB showed the best performance on GABA production when middle copy-number plasmid was used as expression vector in E. coli BW25113. The highest production of GABA reached 308.96 g L(-1) with 99.9 mol% conversion within 12 h, when E. coli ΔgabAB (pRB-lgadB) concentrated to an OD600 of 15 in 3 M l-Glu at 45 °C. Furthermore, the strain could be reused at least three cycles in 2 M crude l-Glu with an average productivity of 40.94 g L(-1) h(-1). The total GABA yield reached 614.15 g L(-1) with a molar yield over 99 %, which represented the highest GABA production ever reported. The whole-cell bioconversion system allowed us to achieve a promising cost-effective resource for GABA in industrial application.

Keywords: Bioconversion; Escherichia coli; Gamma-aminobutyric acid; Glutamate decarboxylase; Whole-cell biocatalyst.

Fig. 1

Time profiles of GABA produced in a MSG buffer and b l-Glu solution by E. coli BW25113 expressing different GadB. Conversions were performed at 37 °C and 25 g wet cell per liter. Data are presented as the mean ± SD values from three independent experiments. Square, down triangle, diamond and circle symbolize the E. coli BW25113 and recombinant E. coli BW25113 harboring pYB-pgadB, pYB-bgadB and pYB-lgadB, respectively

Fig. 2

Production of GABA by recombinant E. coli harboring different plasmids. Square, circle, down triangle, diamond, hexagon and up triangle symbolize the recombinant E. coli harboring pRB-lgadB, pYB-lgadB, pDB-lgadB, pUB-lgadB, pAB-lgadB and pSB-lgadB, respectively

Fig. 3

Effects of reaction condition on GABA production. Conversion reactions were performed at different a temperature and b cell concentrations with shaking at 200 rpm for 3 h. Data are presented as the mean ± SD values from three independent experiments. The bars represent GABA concentrations and the square symbols represent a volumetric productivity, b cell productivity

Fig. 4

Time profiles of a GABA and b residual l-Glu concentration during conversion in different l-Glu concentrations. Conversions were performed at 45 °C and 12.5 g wet cell per liter (OD₆₀₀ of 15). Data are presented as the mean ± SD values from three independent experiments. Square, circle, up triangle, down triangle, diamond, left triangle and right triangle represent the concentrations of l-Glu: 1, 1.5, 2, 2.5, 3, 3.5 and 4 M, respectively

Fig. 5

Time profiles of GABA concentration were obtained using various strains. Diamond, square, up triangle, circle and down triangle represent the recombinant E. coli ΔgabT, E. coli BW25113, E. coli ΔgadB, E. coli ΔgadA and E. coli ΔgadAB harboring pRB-lgadB, respectively

Fig. 6

Time profiles of GABA synthesis from a 3 M and b 2 M crude l-Glu solution. a Time course where diamond, square and circle represent l-Glu concentration, GABA production and pH, respectively. b Repeated use of cells where square, circle and diamond represent cycle 1, cycle 2 and cycle 3, respectively; filled shapes GABA concentrations; open shapes residual l-Glu concentrations