Construction Of The Engineered Strain For Effective Bioconversion Of Glutamic Acid To Gama-aminobutyric Acid

?-aminobutyric acid(GABA)is a non-protein amino acid,which severs as an important inhibitory neurotransmitter in mammalian nervous systems.GABA has several physiological functions,such as blood-pressure decreasion,treatment of epilepsy,anti-anxiety and so on.Thus GABA has been applied extensively in pharmaceuticals,food and chemical industry.This study based on an original metabolically engineered Escherichia coli(BW/pRB-IgadB)with high production of GABA.In order to improve the soluble expression of the heterologous glutamate decarboylase(GAD),the arginine codons and the molecular chaperones were introduced in BW/pRB-IgadB to reduce the translation speed and help the GAD folding.According to the result,the higher catalytic efficiency was achieved by the recombinant strain BW/pRB-IgadBC1,which resulted in the production of 296.12±2.01 g/L GABA with a conversion of 96.0 mol%.The GABA volumetric productivity was 40.45 g/L/h,which improved by 32.6%compared with the original strain.Homologous recombination and site-directed mutagenesis were performed to delete the C-terminal residues of GAD and glutamate-GABA antiporter(GadC).The results of whole-cell bioconversion showed that C-terminally truncated GadC could enhance the GABA/Glu transportation capability of recombinant strain BW(? C)/pRB-IgadB.The GABA production reached 296.63 ± 3.29 g/L with 96.0 mol%conversion by recombinant strain BW(? C)/pRB-IgadB.The GABA volumetric productivity was 42.38 g/L/h with 13.7%improvement compared with the original strain.Furthermore,recombinant strain BW/pRB-IgadB(M4)was constructed by four sites specific mutagenesis of GAD.The whole-cell bioconversion of strain BW/pRB-IgadB(M4)showed that the production of GABA reached 306.65 ±2.73 g/L with 99.1 mol%conversion within 7 hours.Moreover,the Glu residual concentration of system was only 1.45±0.07 g/L,and the GABA volumetric productivity was improved to 43.81 g/L/h with 18.7%enhancement compared with the original strain.To further improve the production of GABA,the methods above were combined to construct recombinant strain BW(? C)/pRB-IgadB(M4C).The highest production of GABA(307.40 ±3.18 g/L)was achieved with 99.4 mol%conversion within 5 hours during the whole-cell bioconversion.The GABA volumetric productivity reached 61.48 g/L/h,which was 0.66-fold higher than the original strain.Finally,the whole-cell bioconversion of strain BW(? C)/pRB-IgadB(M4C)was scaled up in a 5 L reactor,and GABA production reached 308.26± 1.88 g/L with 99.6 mol%conversion within 7 hours.What is more,the GABA volumetric productivity was 44.04 g/L/h with 63.8%improvement compared with the original strain.