Study On Cloning And Expression Of Key Enzyme Genes Of E.Coli Ethanol Production

The production of biomass ethanol is of great significance to solve the problem of petroleum resource shortage and air pollution which are increasingly serious in today's society.Pyruvate formate lyase,acetaldehyde dehydrogenase,ethanol dehydrogenase and other key enzymes have an important effect on the production of ethanol by microbial fermentation,and the expression of key genes in microorganisms affects the enzyme activity and amount of these enzymes.In this study,starting from the pyruvate formate lyase gene and aldol dehydrogenase gene in Escherichia coli K-12,the pyruvate formate lyase gene and aldol dehydrogenase gene were synthesized by solid phase phosphosamide triester method.The pET-PFLB-ADHE plasmid was synthesized by molecular cloning method.The recombinant plasmid was transfected into Escherichia coli BL21(DE3),and a strain of Escherichia coli producing ethanol was obtained.The fermentation conditions,technological parameters and enzymatic properties of the enzyme were systematically studied.The main results were as follows:1.The pyruvate formate lyase gene and aldol dehydrogenase gene were cloned by gene synthesis technology,and the two genes were linked to the plasmid pUC19.The target genes were recovered by double enzyme digestion and agarose gel recovery after being transferred into Escherichia coli DH5? for expanded culture.Then T4DNA ligase was applied to the pET44c+prokaryotic expression plasmid recovered by double digestion,and pET-ADHE plasmid,pET-PFLB plasmid and pET-PFLB-ADHE plasmid were obtained.2.Using pET44c+as the expression vector of Escherichia coli pyruvate formate lyase and aldol dehydrogenase gene,the recombinant plasmid was identified by resistance screening,colony PCR and recombinant plasmid PCR verification.The recombinant pET44c+plasmid with target gene was transformed into Escherichia coli BL21(DE3).SDS-PAGE protein electrophoresis detected 85kD and 96kD protein bands,which were consistent with the expected molecular weight of the target protein,and proved that the gene insertion was correct.3.Using recombinant Escherichia coli as the fermentation strain,through the single factor experiment of five factors and five levels,the process parameters of ethanol fermentation were optimized as follows:pH 8.0,C/N ratio 5:3,shaking table speed 200rpm,strain inoculation amount 5%,IPTG inducer concentration 0.4mmol/L.In response surface design,glucose was added to the fermentation medium to a final concentration of 25g/L as a carbon source,and ethanol yield was 11.98 g/L,reaching 93.96%of the theoretical yield.4.The resistance of the recombinant Escherichia coli to ethanol was studied.The maximum resistance of the recombinant Escherichia coli to ethanol reached 39.28g/L under the culture conditions of pH 8.0,IPTG inducer concentration 0.4 mmol/L,strain inoculation amount 5%,and glucose supplemental fermentation(1.5g/h).5.The enzymatic properties of pyruvate formate lyase and aldol dehydrogenase produced by recombinant Escherichia coli fermentation were studied.LB medium was used as the culture medium,and then anaerobic culture was used for 12h to prepare crude enzyme solution.The optimum reaction temperature of pyruvate kinase was 35?,and the optimum pH was 8.0.The optimum reaction temperature and pH of aldol dehydrogenase were 35? and 8.0.The Km value of pyruvate formate lyase was 1.94 mmol/L and the Km value of aldol dehydrogenase was 1.67mmol/L.The activity of pyruvate formate lyase was promoted by 1 mmol/L Fe2+metal ion.