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. 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. 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.

5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of l-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of l-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than l-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient l-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using l-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose. Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36 promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid. Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.

In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.