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CLONING OF ALCOHOL DEHYDROGENASEII GENE AND PYRUVATE DECARBOXY LASE GENE FROM Zymomonas Mobilis AND EXPRESSION IN Escherichia Coli

Posted on:2003-10-05Degree:MasterType:Thesis
Country:ChinaCandidate:J LuFull Text:PDF
GTID:2120360062490427Subject:Microbiology
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Due to dwindling of fossil fuel, fuel ethanol has been trusted as an alternate fuel for the future.Lignocellulose materials are the most abundant renewable resource on earth. The research for the utilization of lignocellulose. especially for the conversion of lignocellulose into fuel ethanol by microorganisms is being studied intensively.The microorganism which produce ethanol from lignocellulose must be able to dissimilate both five-carbon and six-carbon sugars and consist of the PDC and ADH II which are key enzymes in ethanol formation. Regretfully, no naturally occurring organism can efficiently1 produce ethanol from all monosaccharides. E.coli is capable of metabolizing both hexose and pentose sugars, but it lacks PDC-ADH II system.However,, Zymomonas mobilis is able to produce ethanol efficiently by an overactive PDC-ADH II system, but its utilizable substrate range is restricted to glucose and fructose.The genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase II (ADH II) were amplified from total DNA of Zymomonas mobilis by PCR. The genes of adhB andpdc were inserted into expression vector pSE380 and then transformed into E.coli DH5a. The recombinant strains were induced to express ADH II and PDC with IPTG. .The ADH II and PDC activities were detected by aldehyde indicator plates. SDS-PAGE showed that the relative molecular weights of the adhB and pdc expression product were about 40KD and 60KD respectively.The recombinant plasmid for the production of ethanol was constructed by combining pdc and adhB with ribosomal binding sites and was placed under the control of trc promoter. This plasmid was transformed into E.coli DH5a. Recombinant strains produced 0.08% ethanol (g/g) from 10% glucose under the condition of 150rpm, 37癈 and 72 hours. However, the wild-type E.coli DH5a produced scarcely ethanol.This research successfully introduce the novel pathway for fermenting glucose to ethanol in E.coli by expressing Zymomonas mobilis genes encoding PDC and ADH II, which was reported for the first time.
Keywords/Search Tags:fuel alcohol, alcohol dehydrogenase, pyruvate decarboxylase, cloning, E.coli DH5α, induce expression
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