Reconstruction Of The Glutamate Decarboxylase System In Escherichia Coli For Biosynthesis Of γ-aminobutyric Acid

Gama-aminobutyric acid（GABA）is an important suppressive neurotransmitter in mammalian brain,which has been widley used in food,pharmaceutical,and chemical industries due to its special physiological roles such as anti-hypertension,anti-depression,and blood ammonia reduction.At present,GABA is mainly synthesized by acid-resistant bacteria such as Escherichia coli,Bacillus subtilis,and lactic acid bacteria.The key enzymes responsible for the synthesis of GABA including the pyridoxal-5’-phosphate（PLP）dependent glutamate decarboxylase（Gad B or Gad A）that catalyzes the irreversibleα-decarboxylation of L-Glu to GABA and CO₂,and a cognate membrane bound antiporter Gad C that exchanges the intracellular GABA with extracellular L-Glu.Therefore,characterization of strains with high GAD activity and establishment of an efficient catalytic conversion reaction system is a prerequisite for improving the synthesis of GABA.In this study,using E.coli as a platform,the efficiency of GABA production was substantially improved by reconstructing the GAD system of E.coli on the basis of metabolic engineering,protein engineering,and morphology engineering techniques.（1）The Gad B from Lactiplantibacillus plantarum Yll.03,a GABA producing strain,was expressed in E.coli BL21（DE3）.The Gad B exhibited maximal activity at 40oC and p H 5.0,with the K_m value and catalytic efficiency(k_cat/K_m)of 22.33 m M and 1.04 m M^-1s^-1,respectively,and the specific enzyme activity was 27.79 U/mg.Moreover,the GAD activity was slightly enhanced by Ca²⁺,Zn²⁺,and Mg²⁺at the investigated concentrations.Subsequently,the C-terminal of Gad B was directly truncated to obtain the mutant Gad B_ΔC11 with the highest catalytic activity at near-neutral p H values.Notably,it still had activity when the p H value increased to 6.5 and 7.0,with the catalytic activities of 5.06 U/mg and 2.16 U/mg,respectively.（2）For the tunable expression of the GAD system in E.coli,a double-promoter expression vector p ET28a-ara C-P_BADwas designed and constructed.Then,the gad C or gad C_?C41 of E.coli and gad B_ΔC11 of L.plantarum were successively inserted into the p ET28a-ara C-P_BAD to produce p TA01L and p TA02L,respectively.The expression of gad C or gad C_?C41 and gad B_ΔC11 was under the control of P_T7lac and P_BAD,respectively.The results showed that overexpression of Gad C or Gad C?_C41 by addition of IPTG with different concentrations affected the cell growth.The cell-bound GAD activity for E.coli BL21（DE3）/p TA02L increased·to 489.24±33.01 U/g DCW under the induction of IPTG（12.5μM）and L-arabinose（4 g/L）,which was 1.29-fold higher than that of E.coli BL21（DE3）/p TA01L.These findings suggested that broadening the active p H range of Gad B and Gad C towards neutral p H and employing a tunable expression host for membrane protein production is a promising strategy for high level GABA biosynthesis in E.coli.（3）To further minimize the toxic effects of Gad C_ΔC41 production,the p TA02L was further transferred into E.coli Lemo21（DE3）to produce E.coli Lemo21（DE3）/p TA02L.The results showed that the E.coli Lemo21（DE3）was able to minimize the toxic effects caused by the overexpression of membrane-bound Gad C_ΔC41.Meanwhile,the Lemo21（DE3）/p TA02L was induced with IPTG and L-arabinose and the cells were collected.Then,the cell pellets were re-suspended in200 m L of deionized water with an OD₆₀₀ of 20.L-Glu（88.3 g）was added to start the decarboxylation reaction at 37oC and 150 rpm under un-controlled p H condition.After 5 h,the GABA level reached to a concentration of 279.31±8.12 g/L,which was 1.12-fold higher than that of E.coli BL21（DE3）/p TA02L.（4）To weaken the membrane barrier of engineered strains,an expression vector harboring lac I-P_T7lac,ara C-P_BADand rha RS-P_{rha BAD} promoters was constructed.Then,for the tunable expression of Sul A in E.coli,a plasmid p TASR03L was constructed.Compared with normal cells（CK）,after overexpression of Sul A,the cells became thinner and filamentous,resulting in the increase of cell size and permeability.At the above catalytic system,when the reaction time was extended to 5 h,the GABA production and the space-time yield of E.coli Lemo21（DE3）/p TASR03L were 300.36 g/L g/L and 120.33 g/L/h,reapectively,higher than that observed in E.coli BL21（DE3）/p TASR03L（290.10 g/L and 110.04 g/L/h）.