Abstract
BackgroundGamma-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.ResultsRecombinant 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.ConclusionRecombinant 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.
Highlights
Gamma-aminobutylate (GABA) is an important chemical in pharmacetucal field and chemical industry
E. coli GadB mutant (Glu89Gln/Δ452-466) having catalytic activity in broadened pH range up to pH 7 was developed by engineering E. coli GadB [20], which might allow us to achieve enhanced production of GABA since culture conditions for cell growth and synthesis of L-glutamate, the direct precursor of GABA, can be optimized for GABA production in broadened culture pH conditions
Recombinant C. glutamicum harboring pHGmut successfully produced GABA to the concentration of 5.89 ± 0.35 g/L, but recombinant C. glutamicum harboring pHGwt produced much lower GABA to the concentration of 0.34 ± 0.26 g/L (Figure 1B). It is well-known that C. glutamicum expressing wild type gadB genes could produce GABA when they were cultivated at low pH conditions [13,14]
Summary
Gamma-aminobutylate (GABA) is an important chemical in pharmacetucal field and chemical industry. C. glutamicum expressing E. coli glutamate decarboxylase, which was further engineered by the deletion of the pknG gene encoding serine/threonine protein kinase G, resulted in the production of 31 g/L of GABA in 120 h fermentation [11]. In these studies, wild-type glutamate decarboxylases that have optimal pH around 4.5 for their decarboxylation of glutamate were employed for the production of GABA, but the optimal culture pH of C. glutamicum strain for its growth was much higher than 4.5. GABA was produced and this was the main reason for relatively long time cultivation over 120 h, which resulted in low GABA productivity
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