Metabolic Engineering Of An Industrial Ethanol Producing Yeast To Improve Its Fermentation Performance

As the reserves of petroleum decrease, ethanol, which is renewable, has become the focus of many countries as an alternative liquid fuels to gasoline. Improvement of the fermentation competence of yeast strain by gene engineering and metabolic engineering will decrease the costs of ethanol production. In the present study, combined Cre/loxp system with rDNA site homologous recombination, we have conducted research on industrial ethanol-producing yeast from three respects to establish the multi-gene modification strategy suitable used in industrial ethanol-producing yeast.First, since glycerol is a main by-product consuming up to 4%～10% of the carbon source in industrial ethanol fermentation, to reduce the production of glycerol and lead carbon source flux towards the synthesis of ethanol is an important way to improve the ethanol yield. Thus, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of Saccharomyces cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehydes-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the obtained GPD1 deleted mutant. And then, trehalose was over-synthesized in above recombinant strain by expression of trehalose synthesis genes TPS1 and TPS2 by using the Cre/loxp system. The resultant recombinant strain AG1A1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2) exhibited a 76.0±0.2% (relative to the amount of substrate consumed) decrease in glycerol production and a 8.9±0.1% (relative to the amount of substrate consumed) increase in ethanol yield compared with the parent strain. Besides, the maximum specific growth rate (μmax) and fermentation ability of this yeast recombinant strain were indistinguishable as compared to parent strain in anaerobic batch fermentations.Second, although the materials used for ethanol production contain relatively high protein content (corn: 10-13%; feeding barley: 15-18%), the yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. To improve the substrate utilization and ethanol yield, the gene PEP4, encoding a vacuolar aspartyl protease in S. cerevisiae, and the gene Asp, encoding aspartic protease in Neurospora crassa, was cloned and expressed in industrial ethanol yeast, respectively. The obtained two recombinant strains APB2 (P_PGK1-PEP4-AG1), SA3 (P_PGK1-Asp-AG1) were studied under ethanol fermentation conditions in corn mash fermentations. The ethanol yields of APB2 and SA3 increased by 6.5±0.2% and 5.7±0.1% (relative to the amount of substrate consumed) compared with parent strain. The recombinant strains APB2 and AS3 produced 126.0 g/L and 125.0 g/L ethanol, while the parent strain produced 118.2 g/L ethanol at the end of fermentation. Since the optimal reactive temperature and pH value of PEP4 was coincided with that of SSF (Simultaneous Saccharification and Fermentation), it was chosen and expressed in the recombinant strain AG1A1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2) whose G418 resistance gene was deleted by Cre/loxp system. The resultant strain was named AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1). Third, cellobiose is one of the sugars that cannot be effectively used by S. cerevisiae in the distillate of ethanol fermentation. To engineer the yeast with the ability of assimilation of cellobiose will improve the substrate utilization efficiency and promote the development of cellulose-ethanol fermentation. In our study, the BGL1 gene, encodingβ-glucosidase in Saccharomycopsis fibuligera, was expressed in industrial ethanol-producing strain of S. cerevisiae in three different patterns—in vivo, in vitro and on cell surface. The obtained recombinant strains SBA1 (P_PGK1-BGL1) expression of intracellularβ-glucosidase, SBB1 (P_PGK1-αF-BGL1) expression of extracellularβ-glucosidase and SBC2 (P_PGK1-αF-BGL1-AG1) expression of cell-wall anchoredβ-glucosidase were studied under aerobic and anaerobic conditions in medium supplemented with cellobiose. The results indicated that the parent S. cerevisiae used in industrial ethanol production is likely deficient in cellobiose transporter. However, when theβ-glucoside permease andβ-glucosidase were co-expressed in this strain, it began to uptake cellobiose and the overall performance of this strain in cellobiose-fermentation was improved. The recombinant strain consumed 10 g/L cellobiose and produced 4.4 g/L ethanol in 96 h. After the G418 resistance gene of the recombinant strain AGS1 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1) was deleted by Cre/loxp system, theβ-glucoside permease andβ-glucosidase were co-expressed in this strain and the resultant strain AGPB3 (gpd1△::P_PGK1-gapN, P_PGK1-TPS1-TPS2-PEP4-AG1-bglP-BGL1) was studied in cassava mash fermentations. Not only the recombinant strain AGPB3 can use cellobiose to produce ethanol, but also this strain showed fast growth rate and glucose consumption rate, as well as ethanol production rate. Finally, the recombinant strain AGPB3 exhibited a 76.8% (relative to the amount of substrate consumed) decrease in glycerol production. The ethanol yield was increased from 118.5 g/L to 129.3 g/L, corresponding to 97% of theoretical yield and a 7.5% (relative to the amount of substrate consumed) increase in ethanol yield was achieved compared with the parent strain.