Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range

Abstract

Background: Gamma-aminobutylate (GABA) is an important chemical in pharmacetucal field and chemical industry. GABA has mostly been produced in lactic acid bacteria by adding L-glutamate to the culture medium since L-glutamate can be converted into GABA by inherent L-glutamate decarboxylase. Recently, GABA has gained much attention for the application as a major building block for the synthesis of 2-pyrrolidone and biodegradable polyamide nylon 4, which opens its application area in the industrial biotechnology. Therefore, Corynebacterium glutamicum, the major L-glutamate producing microorganism, has been engineered to achieve direct fermentative production of GABA from glucose, but their productivity was rather low.

Results: Recombinant C. glutamicum strains were developed for enhanced production of GABA from glucose by expressing Escherichia coli glutamate decarboxylase (GAD) mutant, which is active in expanded pH range. Synthetic PH36, PI16, and PL26 promoters, which have different promoter strengths in C. glutamicum, were examined for the expression of E. coli GAD mutant. C. glutamicum expressing E. coli GAD mutant under the strong PH36 promoter could produce GABA to the concentration of 5.89±0.35 g/L in GP1 medium at pH 7.0, which is 17-fold higher than that obtained by C. glutamicum expressing wild-type E. coli GAD in the same condition (0.34±0.26 g/L). Fed-bath culture of C. glutamicum expressing E. coli GAD mutant in GP1 medium containing 50 μg/L of biotin at pH 6, culture condition of which was optimized in flask cultures, resulted in the highest GABA concentration of 38.6±0.85 g/L with the productivity of 0.536 g/L/h.

Conclusion: Recombinant C. glutamicum strains developed in this study should be useful for the direct fermentative production of GABA from glucose, which allows us to achieve enhanced production of GABA suitable for its application area in the industrial biotechnology.

Figure 1

Comparison of GABA production in C. glutamicum producing wild type GAD or mutant GAD in flaks cultivation. (A) SDS-PAGE analysis of production of wild type GAD and mutant GAD. Lane M, protein size markers (kDa). Lanes 1 and 2, C. glutamicum harboring pCES208. Lanes 3 and 4, C. glutamicum harboring pHGwt. Lanes 5 and 6, C. glutamicum harboring pHGmut. Lanes 1, 3, and 5, total protein fractions. Lanes 2, 4, and 6, soluble protein fractions. The open and closed arrowheads indicate the band of wild type GAD and mutant GAD, respectively. (B) Time profiles of GABA concentration. Diamond and circle symbols indicate C. glutamicum harboring pHGwt and pHGmut, respectively.

Figure 2

Comparison of three different promoters for gene expression and GABA production in C. glutamicum . (A) SDS-PAGE analysis of mutant GAD gene expression under the three synthetic promoters (P_L26, P_I16, or P_H36). Lane M, protein size markers (kDa). Lanes 1 and 2, C. glutamicum harboring pCES208.Lanes 3 and 4, C. glutamicum harboring pLGmut. Lanes 5 and 6, C. glutamicum harboring pIGmut. Lanes 7 and 8, C. glutamicum harboring pHGmut. Lanes 1, 3, 5, and 7, total protein fractions. Lanes 2, 4, 6, and 8, soluble protein fractions. (B) Time course of GABA concentration during flask cultivation of C. glutamicum. Triangle, square, circle and diamond symbols indicate C. glutamicum harboring pLGmut, pIGmut, pHGmut and pHGwt respectively. Each point represents the average of three independent experiments.

Figure 3

Time profiles of cell density and GABA concentrations during flask cultivations with different biotin concentrations. Square, circle, triangle and diamond symbols indicate 1 μg/L, 50 μg/L, 100 μg/L, and 500 μg/L, respectively. (A) Time profiles of cell growth at four different biotin concentration conditions. (B) Open and closed symbols indicate the L-glutamate concentrations and GABA concentrations, respectively. Each point represents the average of three independent experiments. Each point represents the average of three independent experiments.

Figure 4

Effect of pH on cell growth and GABA production in flask cultivation of C. glutamicum . (A) Time profiles of cell growth and pH change at four different pH conditions. Triangle, circle and square symbols indicate the pH 4, pH 5 and pH 6, respectively. The open and closed symbols indicate the cell density and pH change, respectively. (B) Time profiles of GABA concentrations during cultivations. Triangle, and circle and square symbols indicate pH 4, pH 5 and pH 6, respectively. Each point represents the average of three independent experiments.

Figure 5

Fed-batch cultivation for GABA production with different pH conditions. (A) Time profiles of cell growth and glucose concentration. Circle, triangle, and square symbols indicate pH 5, pH 6 and pH 7, respectively. Open and closed symbols indicate the glucose concentration and cell density, respectively. (B) Time profiles of L-glutamate and GABA concentrations. Circle, triangle, and square symbols indicate pH 5, pH 6 and pH 7, respectively. Open and closed symbols indicate the GABA and glutamate concentrations, respectively. The first time points for glucose feeding were indicated by gray-solid (pH 5), black-dashed (pH 6) and black-solid (pH 7) arrows.