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A new metabolic route for the production of gamma-aminobutyric acid in Corynebacterium glutamicum

ABSTRACT: Corynebacterium glutamicum shows a great potential for the production of gamma-aminobutyric acid (GABA) from glucose fermentation via putrescine. GABA, a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods and the biodegradable plastic polyamide 4. Here, the effect of GABA in the growth of C. glutamicum was evaluated. It was estimated that the presence 1.1 M of GABA in the medium reduces the maximum growth rate of C. glutamicum to half. It was also shown that the presence of GABA in the medium negatively affects the growth of C. glutamicum in ethanol as sole carbon source. Furthermore, a new route for the production of GABA in C. glutamicum was established. GABA production from glucose fermentation via putrescine was achieved by plasmid-based overexpression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) in a putrescine production strain. The resultant strain can produce 5.3 ± 0.1 g L-1 of GABA. GABA production was improved by avoiding the formation of N-acetylputrescine and by reducing the amount of nitrogen in CGXII medium. Deletion of the genes responsible for GABA catabolism and GABA re-uptake led to an increase in the GABA production of 21% achieving a titer 8.0 ± 0.3 g L-1 and an increase in the volumetric productivity of 41% reaching a productivity of 0.31 g L-1 h-1, the highest volumetric productivity achieved so far for GABA production in C. glutamicum from glucose fermentation in flasks fermentations. The results obtained hitherto are very promising and competitive compared to the traditional pathway for the production of GABA.

ORGANISM(S): Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032

PROVIDER: GSE74258 | GEO | 2015/10/22

SECONDARY ACCESSION(S): PRJNA299574

REPOSITORIES: GEO

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Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 Â± 0.03 gg-1 and a volumetric productivity of 0.04 Â± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.

Project description:Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels. Experiment Overall Design: P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of L-glutamate alone or with the addition of putrescine, agmatine or GABA at 20 mM. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Only genes showing consistent expression profiles in duplicates were selected for further analysis.

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A new metabolic route for the production of gamma-aminobutyric acid in Corynebacterium glutamicum

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