Heterologous expression of cellobiose transporter LacY in Saccharomyces cerevisiae and development of engineered S. cerevisiae for cellobiose utilization

Abstract:

[Objective] A metabolic pathway was designed in Saccharomyces cerevisiae so that it could produce ethanol from cellobiose, a hydrolysate of cellulose. [Methods] First, with the total DNA of Escherichia coli DH5α as template, the LacY gene encoding lactose permease was cloned. With plasmid YEplac181 as the vector, the strong promoter PGK1p of S. cerevisiae was added in front of the LacY, and the CYC1t terminator was added behind LacY to construct the plasmid YEplac181-PGK1p-LacY-CYC1t. Then, the cellobiose transporter (LacY) expression plasmid and β-glucosidase expression plasmid pRS316-PGK1p-gh1-1-CYC1t were successively transferred into wild-type S. cerevisiae W303-1A. The engineered S. cerevisiae W303-1A GL was constructed by heterologous expression of cellobiose transporter LacY and β-glucosidase GH1-1 in W303-1A. Finally, the cellobiose utilization and ethanol yield of W303-1A GL were determined based on fermentation, and the cellobiose activity in the cellobiose metabolic pathway was detected. [Results] W303-1A GL which expressed LacY and GH1-1 was developed, which can use cellobiose to produce ethanol. The ethanol yield of W303-1A GL was up to 3.25 g/L at 24 h, and the production rate was 0.325 g ethanol/g cellobiose, 64% of the theoretical production rate from glucose (0.511 g ethanol/g cellobiose). The highest cell density was OD₆₀₀=10.84 at 54 h, and the activity of intracellular β-glucosidase peaked (0.51 U/mg) at 72 h. [Conclusion] W303-1A GL which can effectively utilize cellobiose is developed. This study is expected to serve as a reference for improving the production of cellulosic ethanol at lower cost.