Ethanol Production By Saccharomyces Cerevisiae With Glycerol As The Carbon Sourse

As a limited nonrenewable resource, fosil fuels will finally be depleted due to the continuous and vast exploitation of humanbeings. The development of technologies for bioethonal production offers an alternative energy form to completely resolve the fosil energy crisis. Ethanol production from starch- or sugar-based agriculture products raises the concern over food safty, while the global grain price is increasingly high. The process of biodiesel production generates about 10% (w/w) glycerol that resultes in serious problems in process economy and environmental protection. One of the possible applications for this excess glycerol is to use it as a carbon source for microorganisms to produce high value-added commodities.Both food crisis and overproduction of glycerol can be solved when glycerol is used as the substrate to produce bioethonal. Our laboratory has constructed a strain LGE4 that carrys deletions in the two functional redundant gene, GPD1 and GPD2, encoding the NAD+-dependent glycerol-3-phosphate dehydrogenase. In this strain glycerol synthesis during ethanol production with glycerol as the carbon source is blocked. This forms the bases of this thesis research aimed at production of bioethanol using glycerol as the substrate.Due to glycerol production is blocked in strain LGE4, excess NADH could not be consumed in such a strain. Therefore, redox ballance is destroyed and the growth rate of LGE4 is negatively affected. A water-forming NADH oxidase, encoded by the noxE gene found in Lactococcus lactis, can oxidize NADH to NAD+ while turn O2 into H2O. So we constructed a strain LGE4-N which express noxE gene in the strain LGE4 to improve its growth.We overexpressed GCY1 encoding glycerol dehydrogenase and DAK1,DAK2 encoding dihydroxyacetone kinase respectively, each of which catalyse the metabolism from glycerol to dihydroxyacetone phosphate in the strain LGE4-N, which is the most important step of glycerol dissimilation. First of all, we constructed plasmids on YCplac33 and YEplac195 to overexpress GCY1,DAK1,DAK2 under the PGK1 promotor, then plasmids were transformed to the strain LGE4-N with noxE overexpressed. Plasmids YCplac33 and YEplac195 were also transformed into the same strain as control. By analysis of the growth performance of these transformants in the medium with glycerol as sole carbon resource, we concluded that overexpression of GCY1,DAK1 and DAK2 in strain LGE4-N positively affected the growth of yeast cells in glycerol-containing medium, and verified the feasibility of our strategy for the construction of recombinant yeast strains that can concert glycerol to ethanol.