Metabolic Engineering Of Escherichia Coil K12 For Improving Gamma-Aminobutyricacid Production

γ-aminobutyric acid(GABA)is a non-protein amino acid widely existing in nature.It plays a role of inhibiting neural transmission in neurotransmitter signals and can be applied in food,medicine and feed industries.GadB is a kind of pyridoxal phosphate(PLP)dependent enzyme,which can catalyze the production of GABA by L-Glu or L-MSG.Recently,there are numerous papers about GABA,but the coenzyme factor PLP synthase gene is constructed on plasmids,which has been rarely reported at home and abroad.The research of this subject is as follows:(1)Plasmid construction:the original strain was E.coliK12ΔgabTΔgabPΔpuuE.After preliminary fermentation,the 10g/L L-MSG culture medium produced 0.21g/L GABA,which increased the yield by 430%compared with the original strain E.coliK12 by 0.05g/L.In the second step,the plasmid was constructed by gadB,SNO1 and SNZ1.The plasmid pTrc99a-gadB and pTrc99a-gadB-SNO1-SNZ1 were constructed successfully through genetic engineering.(2)Construction of two recombinant strains and optimization of fermentation:two plasmids were introduced into the competent cells,and we obtained two strains E.coliK12ΔgabTΔgabPΔpuuE/pTrc99a-gadB and E.coliK12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1.The strain with gadB was fermented in the 10g/L L-MSG,the maximum GABA was 4.6g/L,and the biological conversion rate was 76.6%.The strain with gadB,SNO1,SNZ1 was fermented under the same condition,and the maximum yield of GABA was 4.2g/L.The maximum GABA yield and bioconversion rate were measured using by five L-MSG concentration,respectively.Under conditions of 5g/L,10g/L and 20g/L of L-MSG,The strains E.coliK12ΔgabTΔgabPΔpuuE/pTrc99a-gadB bioconversion rate could be more than 76.3%and the strains E.coliK12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1 only have 61.6%.The strain was expanded with 1L shaker had the 9.4g/L yield of GABA and bioconversion rate was 76.7%.(3)Purification and enzymatic property research:the pure GadB protein was obtained by the DEAE-52 cellulose ion exchange chromatography with preliminary separation by ammonium sulfate salting method,and the SDS-PAGE electrophoresis analysis was performed.The molecular weight was about 53Kda.Enzyme properties showed that the optimal enzyme reaction temperature is 37 0℃;he optimal reaction pH is 4.It was found that the enzyme had good stability at the optimal site and pH.Metal ions had different effects on the activity of GadB enzyme,among which Ba2+ and K+two metal ions had basically no effect on the activity of GadB enzyme,Na+ ions had no significant effect on the activity of enzyme,and decarboxylase activity fluctuated in 96%.The inhibition of metal ion Ni2+ was strong,and the enzyme activity decreased to 80%.