Metabolic Engineering Of γ-aminobutyric Acid Transport System And Decomposition Pathway In Recombinant Corynebacterium Glutamicum

In microorganisms, metabolism of γ-aminobutyric acid(GABA) consists of synthesis, transportation and decomposition. Biosynthesis of GABA is fulfilled by decarboxylation of L-glutamate by L-glutamate decarboxylase(GAD). The synthesized or obtained GABA can be decomposed into succinate by GABA transaminase encoded by gabT and succinic semialdehyde dehydrogenase under certain condition. Export and uptake of GABA are carried out by L-glutamate/GABA antiporter encoded by gadC and GABA permease encoded by gabP, respectively. To prevent the synthesized GABA from decomposing, metabolic engineering of Corynebacterium glutamicum are performed here. The main research contents and results are as follows:1. The strains SYN3 expressing single GAD gene(gadB2) and SYN4 expressing gadC-gadB2 genes were constructed and fermented in shake flasks. To understand whether GadC acts as an L-glutamate/GABA antiporter in recombinant C. glutamicum, the variation of pH value and the concentration of L-glutamate and GABA during fermentation were investigated. The highest concentration of GABA accumulated in SYN3 and SYN4 was 11.4±0.4 g/L and 5.3±1.9 g/L, respectively, indicating that the activity of GadC transporting L-glutamate and GABA in C. glutamicum should be further investigated. Meanwhile, GABA was found to be decomposed under neutral pH.2. A recombinant C. glutamicum strain SYN200 co-expressing two GAD genes(gadB1 and gadB2) was constructed and fermented in shake flasks. To unravel the effect of pH on GABA synthesis, the variation of pH value and the concentration of L-glutamate and GABA were investigated during fermentation. At 72 h of fermentation,GABA production were 16.8±2.2 g/L and 7.5±0.3 g/L when pH was adjusted to 5.0-5.5 and 7.5-8.0, respectively at 60 h. This result showed that acidic pH was more favorable than neutral pH for GABA accumulation. SYN200 was then fermented in a fermentor to investigate the GABA decomposition under pH 5.0-5.5 and pH 7.5-8.0. GABA concentration decreased from 16.5±0.7 g/L to 12.6±0.9 g/L during 60-72 h when pH was controlled at 7.5-8.0. However, GABA concentration increased from 14.9±0.0 g/L to 17.4±1.2 g/L during 60-72 h when pH was controlled at 5.0-5.5. This result demonstrated that GABA was decomposed under neutral pH.3. The gene gabT and gabP of C. glutamicum were respectively deleted and two deletion strains ΔgabT and ΔgabP were constructed. Then recombinant plasmid pJYW-4-gadB1-gadB2 was transformed into the two deletion mutants, generating two new recombinant strains, i.e. gabT deleted GAD strain SYN201 and gabP deleted GAD strain SYN202. SYN201 and SYN202 were fermented in fermentors to investigate the effect of gabT and gabP deletion on GABA decomposition. GABA concentration of SYN201 decreased from 23.9±1.8 g/L to 17.7±0.7 g/L during 72-84 h when pH was controlled at 7.5-8.0; but GABA concentration of SYN202 remained at 18.6-19.4 g/L. This result demonstrated that deletion of gabP could effectively alleviate GABA decomposition in C. glutamicum, whereas deletion of gabT can’t prevent GABA decomposition.