Surface Display Of Phytase And Pectinesterase On Saccharomyces Cerevisiae And Their Application

Production of bioethanol using starch as raw material has become very prominent technology. However, phytate and/or pectin contained in the raw material not only decrease ethanol production efficiency, but also lead to decline in the value of the distillers dried grains with solubles(DDGS) and an increase in phosphorus discharge. In this study, to decrease phytate and/or pectin content in an ethanol fermentation process, Saccharomyces cerevisiae was engineered for heterologous expression of phytase and pectinesterase on the cell surface individually or simultaneously. The main research results are as below:(1) The phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted into the downstream of the secretion signal gene, to produce a yeast surface-display expression vector p MGK-AG-phy, which was then transformed into S. cerevisiae CICIMY0086. Recombinant yeast strain PHY successfully displayed phytase on the surface of cells with 6.4 U·g-1 wet cells. The recombinant enzyme performed the maximal activity at 55 ℃, p H 4.0, and this enzyme had high activity and great p H stability from p H 3.5 to 4.5. In the simultaneous saccharification and fermentation of corn flour, the biomass of the PHY strain was 4.3×108 cell·m L-1, increased by 7.5% compared to the control; the ethanol production and fermentation efficiency of the PHY strain were 112 g·L-1 and 89.5%, increased by 3.7% compared to the control; moreover, the phytate concentration in dry vinasse was 0.06%(w·w-1), decreased by 91.2% compared to the control, the recombinant PHY improved the utilization value of DDGS and reduced the discharge of phosphorus, which is conducive to the protection of the environment.(2) The pectinesterase gene was fused with the α-agglutinin gene to produce a yeast surface-display expression vector p MGK-AG-PE, which was then transformed into S. cerevisiae CICIMY0086. Recombinant yeast strain PE successfully displayed pectinesterase on the surface of cells with 2.6 U·g-1 wet cells. The recombinant enzyme performed the maximal activity at 60 ℃, p H 5.0, and this enzyme had high activity and great p H stability from p H 4.0 to 5.5. In the simultaneous saccharification and fermentation of sweet potato, the ethanol production and fermentation efficiency of the PHY strain were 95 g·L-1 and 88.1%, increased by 2.2% compared to the control; the viscosity of fermented solution of the PE stain was lower than the parent, the viscosity of the PE stain was 120 m Pa.s and the viscosity of the parent was 145 m Pa.s in the time of fermentation 12 h, decreased by 20.8%, viscosity reduction was benefit to enzyme function and metabolism of S. cerevisiae, can also reduce the burden of equipment, saving the energy consumption.(3) The recombinant yeast strain PP displayed phytase and pectinesterase on the surface was successfully constructed. In the fermentation of corn flour and sweet potato, the biomass of the PP strain were 4.4×108 cell·m L-1 and 4.3×108 cell·m L-1, increased by 10.0% and 7.5% compared to the control; the ethanol production of the PP strain were 113 g·L-1 and 96 g·L-1, increased by 4.6% and 3.2% compared to the control; the viscosity of the PP stain were 84 m Pa.s and 89 m Pa.s in the time of fermentation 24 h, decreased by 7.7% and 15.2% compared to the control; the phytate concentration in dry vinasse were 0.08% and 0.02%(w·w-1), decreased by 88.2% and 80.0% compared to the control. The ethanol production of the recombinant yeast strain PP was higher than the parent S. cerevisiae, and the phytate concentration in dry vinasse and the viscosity of fermented solution were both lower than the parent S. cerevisiae.