Of Pdc And Adh Ii Gene Expression Vector And Its Expression In Escherichia Coli

With the rapidly development of world economy and rapidly growth of population, it makes the demand for various natural resources increase day by day, especially non-renewable resource. Fuel ethanol has been expected to become an alternate fuel for the nature. For the conversion of lignocellulose into fuel ethanol by microorganisms is being studied intensively.Pyruvate decarboxylase and alcohol dehydrogenaseⅡcan convert pyruvate into ethanol. Escherichia coli is capable of metabolizing five-carbon sugars and six-carbon sugars of lignocellulose, but it lacks of pyruvate decarboxylase and alcohol dehydrogenaseⅡ, and the product of ethanol is very low. In order to increase the product of ethanol in the Escherichia coli, this experiment cloned the gene of the pyruvate decarboxylase and alcohol dehydrogenaseⅡof Zymomonas mobilis to the Escherichia coli, construction of recombinant strain of E. coli can not only continue to apply to the production, but also become the host strains of the two target gene.In this experiment, PCR amplified the gene of the pyruvate decarboxylase and alcohol dehydrogenaseⅡof Zymomonas mobilis, and the model is the genomic DNA of Zymomonas mobilis. pdc gene connecting with the adhB gene which contains the RBS was under the control of the T7 promoter, form the polycistronic expressing plasmid pQR-PRA which was transferred into the Escherichia coli BL21(DE). The activity of the pyruvate decarboxylase and alcohol dehydrogenaseⅡcan be tested by the aldehyde indicator plates. The recombinants QR-PRA3 which had the deepest color was chosen for optimizing the enzemy expressing conditions and quantitative detection. The results showed that the best expressing condition is containing IPTG 1.0mmol/L, inducing 7h, 37℃. The highest enzemy activity of the pyruvate decarboxylase is 1.34U/mg, and of the alcohol dehydrogenaseⅡis 3.88U/mg. The SDS-PAGE result showed that the recombinants has the relative molecular weights of the adhB and pdc expression product were about 40kD and 60kD respectively.Recombinant QR-PRA3 was cultured for 72h at 37℃with 5% glucose in LB medium. Its ethanol production was 6.86g/L and its ethanol yield was 0.40g/g. Recombinant QR-PRA3 was cultured for 72h at 37℃with 5% xylose in LB medium. Its ethanol production was 0.77g/L and its ethanol yield was 0.13g/g. Recombinant QR-PRA3 was cultured for 72h at 37℃with 3% glucose and 2% xylose in LB medium. Its ethanol production was 2.27g/L and its ethanol yield was 0.18g/g. Control strains without pQR-PRA did not produce ethanol.This research successfully introduced an ethanol biosynthesis pathway into Escherichia coli by expressing genes pdc and adhB, which set up a base for constructing a stable engineering strain that could ferment decomposition products of lignocellulose materials to fuel alcohol with high efficiency.