Metabolic Engineering Of Escherichia Coil For Gamma-Aminobutyricacid Production

Gamma-aminobutyric acid(GABA)as one of many non-protein amino acids,which can be obtained from plants to microorganisms and mammals,It can serves with potential applications widely in biomedicine,foodstuff,agriculture,and feed industry.The three main preparation methods are microbial fermentation,plant enrichment and chemical synthesis.In this paper,the E.coli K12 wild-type strain was used as a starting strain to improve the metabolic pathway of GABA for enhancing the fermentation production of GABA by genetic engineering.The main results are as follows:1.Three genes,gabT,gabP and puuE,related to the GABA pathway in Escherichia coli K12 were knocked out by Red homologous recombination.The homozygous linear fragment of about 75bp was constructed and the gabT gene was knocked out by the expression of Exo,Beta and Gam from pKD46.And then the plasmid pCP20 encoding the FLP site-specific recombinase was introduced to eliminate the resistance gene at the target gene position.PCR was carried out using the identification primers flanked target gene to obtain the product fragment,the knockout of the gabT gene was identified by the size of fragment and the gene.knockout strain E.coli K12 ?gabT was obtained.Subsequently,E.coli K12 ?gabT was used as the starting strain to knock out its gabP gene successfully,and the knockout strain E.coli K12 ?gabT ?gabP was obtained.Finally,E.coli K12 ?gabT ?gabP was used as the starting strain,and the puuE gene was knocked out to obtain Gene knockout strain E.coli K12 ?gabT?gabP ?puuE.2.The gadA,gadB and gadC genes related to GABA synthesis pathway were cloned into the plasmid ptrc99A,and the recombinant plasmids ptrc99A-gadABC surefire were obtained by double digestion and sequencing.The recombinant plasmid was transferred into the knockout strain E.coli K12 ?gabT ?gabP ?puuE to obtain the engineering strain E.coli K12 ?gabT ?gabP ?puuE/ptrc99A-gadABC.At the shake flask level,the fermentation yields GABA.Compared with the yield 0.11g/L of GABA of the wild-type strain E.coli K12,the yield of GABA of Escherichia coli K12 ?gabT?gabP ?puuE increased to 0.73g g/L,which proved the feasibility of knocking out the three genes to enhance GABA production.The recombinant plasmid was introduced into the gene knockout strain to produce GABA.The yield of GABA was 6.4g g/L,Compared with the gene knockout strain E.coli K12 ?gabT ?gabP ?puuE,which was greatly improved,Further proof of the success of genetic engineering means to transform GABA metabolic pathway.3.The fermentation medium and fermentation conditions of engineering strain E.coli K12 AgabT ?gabP?puuE/ptrc99A-gadABC were optimized.Firstly,single factor experiments were carried out to determine the optimum fermentation medium composition and fermentation conditions.On this basis,the PB test was carried out to select the key factors affecting the GABA production in the fermentation medium,namely the substrate concentration,the glucose concentration and the tryptone concentration.Then,the the results of response surface experiments were showed that GABA yield reached the maximum value of 19.38g/L,when the three key factors reached the optimal value that the concentration of glutamate was 34.59g/L,the concentration of glucose was 8.97g/L and the concentration of tryptone was 24.47g/L.The optimum fermentation medium components were:glucose 8.97 g/L,yeast 16 g/L,tryptone 24.47 g/L,Magnesium sulfate 1.0 g/L,L-glutamic acid 34.59 g/L,ammonium sulfate 5 g/L,phosphate 40mM.The optimum fermentation condions were:inoculation quantity 1%,liquid quantity 50mL(250mL triangle bottle),the optimal pH of GAD enzyme was 4.0,the optimum time to adjust pH was 4h after adding IPTG,the optimum final concentration of IPTG was 1.2mM,which were laided the foundation for the production of GABA via industrial fermentation.