| γ-Aminobutyric acid(GABA) is an amino acid which has several well-characterized physiological functions. In food and pharmaceutical fields, GABA has been used widely. Corynebacterium glutamicum is an important amino acids producer including L-glutamate and L-glutamate is the direct precursor of GABA. By expressing the exogenous gad gene(such as gadB1 and gadB2) which encodes glutamic acid decarboxylase(GAD), C. glutamicum can be engineered to convert its accumulated L-glutamate into GABA. However, under neutral conditions, GABA can be decomposed into succinic acid through two-step reaction catalyzed by GABA transaminase(GABA-T) and succinate semialdehyde dehydrogenase(SSADH). Previous study found that after deleting gabT gene which encodes a GABA-T in recombinant C. glutamicum(ATCC13032ΔgabT/pJYW-4-gadB1-gadB2, written as SYN201), GABA still decomposed under neutral conditions. Therefore, metabolic engineering and decomposition of GABA in recombinant C. glutamicum were researched here. Meanwhile, enzymes’ properties of the relating enzymes involved in GABA decomposition were studied. The main research contents and the results are as follows.(1) The plasmid pJYW-4-gadB1-gadB2 was transformed into an L-glutamate industrial producer, C. glutamicum S9114 to construct a recombinant strain S9114/pJYW-4-gadB1-gadB2. To reduce the formation of by-products L-alanine, alaT which encodes alanine aminotransferase was deleted and two stains, S9114ΔalaT and S9114ΔalaT/pJYW-4-gadB1-gadB2 were constructed. Shake flasks fermentation results showed that the L-alanine production in S9114ΔalaT was only 11.4% lower than that in S9114, and that in S9114ΔalaT/pJYW-4-gadB1-gadB2 was only 5.5% lower than that in S9114/pJYW-4-gadB1-gadB2. RT-PCR showed that the transcription level of avtA gene coding valine aminotransferase increased in S9114ΔalaT/pJYW-4-gadB1-gadB2, making the L-alanine concentration not reduced significantly. Meanwhile, at the later period of fermentation, along with the increase of pH value, GABA decomposition generated.(2) To prevent the GABA decomposition in C. glutamicum, another transaminase gene NCgl2515 was deleted in the gabT-deleted C. glutamicum ATCC13032 strain SYN101 constructed previously and then plasmid pJYW-4-gadB1-gadB2 was transformed, generating the NCgl2515 and gabT double deleted GAD strain SYN203. After fermented in a fermenter, GABA production remained at 25.0 g·L-1 under neutral conditions, indicating that GABA catabolic pathway was blocked in this strain. Therefore, NCgl2515 may involve in GABA decomposition.(3) To realize whether NCgl2515 encodes GABA-T in C. glutamicum, gabT, gabD and NCgl2515 were cloned and over-expressed in Escherichia coli BL21(DE3). Enzymatic properties of GabT, GabD and NCgl2515 protein were analyzed after purified. The results showed that NCgl2515 protein had low GABA-T ativity. But when coupled with GabD, NCgl2515 protein showed GABA-T activity and was able to use both pyruvate and alpha-ketoglutaric acid(α-KG) as the amino acceptor. The pyruvate and α-KG-dependent activity of NCgl2515-GabD proteins were 0.034 and 0.032 U·mg-1, respectively. Therefore, NCgl2515 protein is a new GABA-T. The optimal pH is 7.5-8.0. However, the GABA-T activity of GabT protein is high(1.29U·mg-1), but GabT only uses α-KG as the amino acceptor. The optimum pH of GabT is 7.8 and its activity decreases almost completely when pH is lower than 6.0. Therefore, the two GABA-T i.e. NCgl2515 protein and GabT only catalyze decomposition of GABA under neutral condition. |
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