Fermentation Of Cassava Starch To Ethanol By Genetic Manipulation Of Zymomonas Mobilis

In recent years, we have to recognize that the global rude oil reserve is finite, and its depletion is occurring much faster than previously predicted. In addition, the environmental deterioration resulting from the over-consumption of petroleum-derived products, especially the transportation fuels, is threatening the sustainability of human society. Ethanol, as one of the ideal alternatives, has attracted more and more attention recently. Ethanol is one of the most advanced liquid fuels because it is environmental friendly, which adds octane value to the gasoline blend and provides a cleaner burning. Therefore, ethanol is particularly useful in industrial applications and transportation. It is able to reduce significantly the amount of oil imported and it can replace part of the present fuel.In recent years, fermentative production of ethanol from renewable resources has received attention due to increasing petroleum shortage. For the last three decades, ethanol production by the bacterium Zymomonas mobilis has been studied extensively. Comparative performance trials have suggested that Z. mobilis may become an important fuel ethanol-producing microorganism because of its 5 to 10%higher yield and up to fivefold-higher specific productivity compared with traditional yeast fermentations. Unfortunately, despite these advantages, Z. mobilis is not suitable for the industrial ethanol production.Its substrate spectrum is narrow, since it uses only sucrose, glucose, and fructose as carbon sources (Swings, J.1977). Numerous efforts have been undertaken to broaden its substrate or product range (for reviews and references therein, see references 14,27, and 28).To enhance the foreign gene expression levels in Z.mobilis, the strong native promotor of the pyruvate decarboxylase (Ppdc) in Z.mobilis is necessary for the expression vector. A fragment of a-mating factor isolated from Yeast expression vector pPIC9K by PCR. PCR was performed to generate a 1194 bp fragment of EGIII from the cDNA of Trichoderma viride as a template. PCR was performed to generate a 1851 bp fragment of GA I from the cDNA of aspergillus awamori as a template.In order to improve Z.mobilis substrate utilization substrate range, three recombinant plasmid pBPG, pBPE and pBPGE were constructed. Transformation of Z. mobilis via electroporation,we got the recombinant bacterias Z.mobilis ATCC31821 (pBPSG), Z.mobilis ATCC31821 (pBPSE) and Z.mobilis ATCC31821 (pBPSGE).Their activity are 0.94U/ml,0.76U/ml and 1.57U/ml, Z.mobilis, Z.mobilis (pBBR1-MCS2) activity was not detected.Fermentation of recombinant strains of Z.mobilis, Z.mobilis (pBBR1-MCS2), Z.mobilis ATCC31821 (pBPSG), Z.mobilis ATCC31821 (pBPSE) and Z.mobilis ATCC31821 (pBPSGE) using cassava. Z. mobilis yield reached the maximum at 48h, Z.mobilis ATCC31821 yield 9.05°, Z.mobilis ATCC31821 (pBBR1-MCS2) yield 9.02°, Z.mobilis ATCC31821 (pBPSG) yield 9.34°, Z.mobilis ATCC31821 (pBPSE) yield 9.46°, Z.mobilis ATCC31821 (pBPSGE) yields 9.71°Hence in our study, in order to to improve Zymomonas mobilis substrate utilization range, we have recently developed a strain of Z. mobilis by introducing genes that encode the endoglucanaseⅢ(EGⅢ), glucoamylaseⅠ(GAⅠ) activities necessary for converting cellulose and starch to glucose. So, Z. mobilis could more efficient use of cassva starch.