Genetic Engineering Of Escherichia Coli For Gamma-Aminobutyricacid Production

GABA(γ-aminobutyric acid)is a four carbon non-protein amino acid that is widely distributed in plants,animals and microorganisms.As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid,GABA functions as an inhibitory neurotransmitter in the brain.Synthesis of GABA is catalyzed by glutamate decarboxylase,therefore,the optimal fermentation condition is mainly based on the biochemical properties of the enzyme.As a coenzyme of many enzymes in the amino acid metabolic process,pyridoxal phosphate(PLP)catalyzes a large number of metabolic reactions involving amino acids.therefore,these factors are summarized to provide the most up-dated information for effective GABA synthesis.In the current study,E.coli BL21 was employed to biosynthesize GABA.The GABA productions and productivities were enhanced by genetic engineering modifications,and the optimizations of fermentation conditions.The main work was described as follows:(1)Direct production of GABA from sodium glutamate in recombinant E.coli.In this study,recombinant plasmid pET28a-gadA and pET28a-gadA-SNO1-SNZ1 was constructed.The plasmids were transformed into E.coli BL21(DE3).At the shake flask level,A two-stage pH control strategy was used to ferment with different concentrations of glutamate sodium.E.coli BL21/pET28a-gadA produced 5.541g/L GABA In the MSG fermentation broth with a concentration of 10g/L,and the theoretical yield of GABA reached a maximum of 91%.pET28a-gadA-SNO1-SNZ1 produced 8.454g/L GABA In the MSG fermentation broth with a concentration of 15g/L,and the theoretical yield of GABA reached a maximum of 92.7%.It shows that PLP,which was synthesize by Escherichia coli,can increase the production of GABA and the catalytic efficiency of glutamate decarboxylase.(2)Isolation and purification of recombinant glutamic acid decarboxylase and its enzymological properties:In this study,E.coli BL21/pET28a-gadA-SNO 1-SNZ1 was induced by IPTG.The characteristic proteins were isolated and purified by Ni2+ affinity chromatography and analyzed by SDS-Page electrophoresis.The results showed that the ratio of recombinant glutamic acid decarboxylase activity after purification reached 19U/mg,which was 4.41 times of crude enzyme solution,and the total yield was 12.8%.The purified recombinant GadA’s optimal reaction pH and temperature were 4.5 and 50℃.It indicates that glutamic acid decarboxylase has a strong pH dependence.The enzyme was stable under 40℃ for 150 minutes,however,the GadA lost 82.1%enzyme activity after 70℃ for 150 minutes.The enzymes activity could be inhibited by Ca2+.And Ba2+,Co2+,Mg2+ had no effect on enzyme activity.(3)immobilized Cell of E.coli BL21/pET28a-gadA-SNO1-SNZ1:In this study,we developed an alginate immobilization method to immobilize E.coli BL21/pET28a-gadA-SNO1-SNZ1 cells using bivalent ions(Ca2+and Ba2+)for alginate crosslinking.Balcium alginate beads were not stable under different reaction conditions which might be due to the sensitivity of Ba-alginate beads to higher Salt ions concentration.Sensitivity of Ba-alginate beads against various chemical reagents,such as phosphate,citrate,and non-gelling agents(sodium and magnesiumions).Immobilized cells showed 50%less activity when compared with free cells because of the substrate-product diffusion limitation,which may be one possible reason for this lower activity.The optimal conditions of immobilized cells were optimized.The optimized condition was 30mg/mL wet Escherichia coli was embedded by 2.5%sodium alginate,and hardened these cells for 10 hours with 0.3M calcium chloride solution.Immobilized and free cells had the same optimum pH and reaction temperature,whereas the operational stability of the immobilized cell was threefold higher as compared to free cells at 37℃.The deactivation energy of immobilized cells was higher in comparison to free cells.