Improvement Of γ-aminobutyric Acid Biosynthesis Ability Of Microbial Cells

Glutamate decarboxylase (GAD) could catalyze the a-decarboxylation of glutamate to generate neutral γ-aminobutyric acid (GABA) by consuming H+. GABA is an important inhibitory neurotransmitter in animals and has major physiological functions such as hypotensive agent, diuretic, and tranquilizer, and can be used as a bioactive component in food, pharmaceutical and feed industry. Furthermore, GABA is used as a precursor in the environmentally safe synthesis of a variety of nitrogen-containing industrial chemicals such as polyamide 4. In this research, the cell-bound GAD activity of the GABA-producer Lactobacillus brevis CGMCC 1306 and the recombinant E. coli cells over-expressing GAD (BL21(DE3)-pET28a-gadB) were improved for preparing whole cell biocatalysts with higher GAD activity. In addition, an efficient and economic GABA production method by coupling Glu decarboxylation reaction with lactic acid fermention was also developed. The main results are as follows:1. The GABA biosynthetic ability of Lb. brevis CGMCC NO.1306 were improved by increasing the copy numbers of its key autologous operons related to GABA systhesis. The two operons of GABA systhesis system in Lactobacillus brevis CGMCC NO.1306 (gadRCA operon and gadB operon) were cloned and sequenced firstly, and alignment results showed that both of the sequences of the two operons were 99% with the corresponding sequnences of Lb. brevis ATCC 367. Then the transcriptional levels of the related genes of GABA systhesis system durning the pH-control fermentation process were analyzed by quantitative real-time PCR, The results showed that the changes of transcriptional levels of GAD acid resistance system activator gene (gadR), GAD gene A (gadA) and Glu-GABA antiproter gene (gadC) were positively correlated with the cell-bound GAD activity change, while the change of transcriptional levels of GAD gene B (gadB) did not show any correlation with the cell-bound GAD activity change, so the GABA synthesis ability of Lb. brevis CGMCC NO.1306 was determined by gadRCA operon. By using plasmid pMG36e, the copy numbers of gadRCA operon were increased in Lb. brevis CGMCC NO.1306. The cell-associated GAD activity of the transformants was improved in all fermentation period. At 24 h, the cell-associated GAD activity of the transformants reached the highest (0.86 U/mg), which was 1.23-fold higher than that of original one.2. The cell-associated GAD activity of BL21(DE3)-pET28a-gadB was improved by overexpressing Glu-GABA antiporter (GadC), which can enhance the intracellular interaction of GAD with its substrate by overcoming the cell envelope permeability barrier against the diffusion of substrates (Glu) and products (GABA). The cell-associated GAD activity of the transformants (BL21(DE3)-pET28a-gardBC) was higher by a factor of 2.6 in comparison; however, over-expression of GadC inhibited cell growth severely. The total cell-associated GAD activity per volume of BL21(DE3)-pET28a-gadBC culture was increased by only a factor of 1.39 compared with BL21(DE3)-pET28a-gadB.3. To develop more efficient ways for promoting substrate and product to diffuse through the cell envelope and improving BL21(DE3)-pET28a-gadB cell-associated GAD activity, its cells were permeabilized by either organic solvents, surfactants, cell wall synthesis inhibitor, heat or over-expression membrane-active peptide (Maltose binding protein-Magainin Ⅱ fusion protein, MalE-MagⅡ). (1) Permeabilization with organic solvents increased cell-associated GAD activity as a function of their hydrophobicity, and hexane was the most effective, increasing cell-associated GAD activity by a factor of 9.65 (6.72 U/mg) under the optimum conditions (1% hexane,5 min); (2) Triton X-100 was the most effective among the testing surfactants to enhance cell-associated GAD activity, increasing cell-associated GAD activity by a factor of 10.8 (7.53 U/mg) under the optimum conditions (1% Triton X-100,5 min); (3) Permeabilization with 5 μg/mL ampicillin (cell wall synthesis inhibitor) improved cell-associated GAD activity by a factor of 6.24, but it had an 40.3% inhibitory effect on the yield of cell biomass; (4) Heating BL21(DE3)-pET28a-gadB at 70℃ for 30 min increased cell-associated GAD activity by a factor of 13.1, and GAD did not leak from the permeabilized cells. (5) The permeabilization way for over-expression MalE-Magll could not improve cell-associated GAD activity of BL21(DE3)-pET28a-gadB. Heating BL21 (DE3)-pET28a-gadB at 70℃ for 30 min was the optimum method for increasing cell-associated GAD activity (13.1-fold increase). Moreover, this method can avoid potentially hazardous agent and is more cost-effective and convenient to perform.4. The heat-permeabilized BL21(DE3)-pET28a-gadB cells were immobilized on Ca-alginate gel beads to improve its stability, the biotransformation ability of beads maintained over 60% of their initial ability after 20 consecutive batches under the optimum conditions (cell density 9.28 mg/mL, pH 4.8, temperature 50℃, CaCl2 0.05 M, PLP 0.1 mM), the beads retained 90% of their initial activity after 30 days of storage at 4℃.5. Production of GABA and lactic acid by coupling Glu decarboxylation reaction catalyzed by immobilized permeabilized BL21(DE3)-pET28a-gadB with lactic acid fermentation conducted by Rhizopus oryzae was developed. By coupling the two biochemical processes together and controlling pH between 4.7 and 5.0, a GABA concentration of 44.8 g/L with 96.7% conversion from Glu and a lactic acid concentration of 80.2 g/L with a yield of 67% from glucose were obtained. These data indicated that the two biochemical processes could couple well in one reactor.