Construction Of Genetically Engineered Bacteria E.coli To Produce Ethanol Using The Pentoses And Hexoses

On Earth, Biomass produced by plants through photosynthesis lignocellulose, up114.5billion t a year. The method using acid hydrolysis or enzymatic hydrolysis oflignocellulose into reducing sugars will generate a five-carbon sugar (D-xylose andL-arabinose) and hexoses (glucose, galactose and mannose). Six-carbon sugar about2/3, five-carbon sugar about1/3. Five-carbon sugars can not be fermented to produceethanol by the yeast. If you can find a way to mix the sugar as raw material forethanol production in the engineered bacteria. Ethanol production could increase25%theoretically. Thus reducing production costs.E.coli containing pentose utilization of all the necessary enzymes. However, theproduct formed by fermentation under anaerobic conditions is complex. The ethanolis just one small part of the total metabolites. Ethanol production stage by pyruvatedecarboxylase and alcohol dehydrogenase Ⅱ catalytic completed. E.coli lacking PDC,and intracellular expression of very low levels of ADH Ⅱ, So the final metabolite ofethanol content is very low. Because of E. coli genetic background is very clear, so wecan import pdc and adh II gene into Escherichia coli to built engineering bacteriawhich can using five-carbon sugar fermentation to produce ethanol.In this paper, a method for the synthesis of synthetic genes derived fromZymomonas mobilis pyruvate decarboxylase gene and alcohol dehydrogenase genefrom Escherichia coli. The sequencing results showed that the synthetic adh baseerror rate6.2‰, which accounted for the overall nucleotide deletion error error rateof37.8%, base insertion errors accounted for28.9%of the overall error rate. Pdcsynthetic base error rate error rate4.8‰, which bases delete the wrong overall errorrate of51.4%, the base insertion errors accounted for31.4%of the overall error rate.The synthesis of pdc and adh genes connected to PET-23a plasmid constructPET-AP recombinant vector. Electroporation it into E.coli BL21(DE3) competentcells, After ampicillin resistance screening, The recombinant vector PET-APcontaining pdc and adh genes in successfully transformed into E.coli BL21.Successfully constructed numbered SWZX426engineering bacteria.Assaying results show that with the growth of time training, building ofgenetically engineered bacteria in the adh and pdc enzyme activity also increases. To 10h after training, activity growth slowed, the highest level of ADH activity8.9U/ml, PDC enzyme activity was23.6U/ml.E.coli SWZX426individually use glucose as the sole carbon source fermentationethanol can also be individually utilize xylose fermentation ethanol as the sole carbonsource. Indicate success in the E.coli cells constructed pyruvate decarboxylationpathway, while enhancing the vitality of alcohol dehydrogenase, Construction ofengineering bacteria make ethanol became the major metabolite.By further optimizing the initial pH, temperature, and speed of ventilation, theconstructed E.coli strains could effectively produce ethanol from xylose in6.98g/L and6.05g/L yield. Results of Gas Chromatography analysis to the distilled products fermented from xylose,glucose or mixture of them.