Construction Of A Highly Efficient Synthetic ?-aminobutyric Acid Engineering Strain

?-aminobutyric acid?GABA?,a four-carbon non-proteinogenic amino acid,is widely found in plants,animals,plants and various microorganisms.It plays an important role in the food,feed and chemical industries.In addition,GABA is an important inhibitory neurotransmitter in the human cerebral cortex,hippocampus,and cerebellum.GABA is formed by irreversible?-decarboxylation of glutamate under the action of L-glutamate decarboxylase?GAD?.Screening for highly active glutamate decarboxylase and addressing the acid dependence during the biosynthesis process are of great significance for optimizing engineered strains for industrial application.Based on the previous high-throughput screening of three GAD mutants,GadBM1,GadBM4 and GadBM8 were purified by affinity chromatography,and the enzyme activities of the purified mutants at pH 4.6 and pH 6.0 were determined..The results showed that the enzyme activities of GadBM4 and GadBM1 were significantly increased under two different conditions.At pH 4.6,the k_cat/K_m of GadBM4 and GadBM8 were1.40 times and 1.31 times of the original protein?GadBM0?,respectively.At pH 6.0,the k_cat/K_m of GadBM4 and GadBM1 were 3.51 times and 4.47 times of the original protein,respectively.The above mutants were used to construct the engineered strains,and the GABA synthesis of the engineered strains by whole-cell bioconversion was detected.The results showed that the productivity of the engineered strain BW???/pYBH-LbgadBM4was significantly higher than that of BW???/pYBH-LbgadBM1 and BW(?/pYBH-LbgadBM8.Compared with the starting strain BW???/pYBH-LbgadBM0,the productivity of BW???/pYBH-LbgadBM4 increased by 67.08%.Furthermore,the GABA production of BW???/pYBH-LbgadBM4 reached 289.90±7.11 g/L in 5 hours.Combining the enzymatic data and biocoversion results,we selected the BW???/pYBH-LbgadBM4 engineered strain for subsequent experiments and named it BW???/pYBH-LbgadB*.In the previous study,the Gad???C mutant was obtained by truncating glutamate-GABA antiporter?GadC?,and the transporting ability and pH activity range of the protein were significantly improved.Here,four engineered strains BW???/pYB-LbgadB*-linker-gad???C,BW???/pYB-LbgadB*-gad???C,BW???/pYB-LbgadB*-gadW and BW???/pYB-LbgadB*-gadX.were constructed by overexpressing Gad???C and the two regulator GadW and GadX encoding genes.The function of which is to enhance Gad???C transporting ability by direct or indirect methods.The results of whole-cell biconversion showed that the highest productivity of GABA synthesis by the engineered strain BW???/pYB-LbgadB*-gadX was 8.73%higher than that of the original strain,and the other engineered strains did not achieve significant improvement in catalytic efficiency.The engineered strain BW???/pYB-LbgadB*-gro was constructed by integrating the chaperone protein with the selected mutant GadB*.The results of whole-cell biconversion showed that the highest synthesis productivity of GABA of the engineered strain was three times that of the original strain?72 g/L/h?,reaching 202 g/L/h.SDS-PAGE results showed that the soluble GAD of the engineered strain increased significantly.The SG104?BW25113?poxB::acs?strain which was modified in the acetic acid synthesis pathway was used as a host cell,and the engineered strains SG/pYB-LbgadB*and SG/pYB-LbgadB*-gro were constructed.The results of whole-cell biconversion showed that the average productivity of GABA synthesis in the two strains were 64.82 g/L/h and 73.87 g/L/h,respectively,which was 10.50%and 38.00%higher than that of BW???as a host cell.The GABA production of SG/pYB-LbgadB*was 300.89 g/L±7.19 g/L during 7 hours by whole-cell biconversion,and the Glu residue was only about 5 g/L.