Identification of Gene Combinations for CO ₂ -fixation Pathway Construction and Characterization of Fermentation Phenotypes in Saccharomyces cerevisiae

Reducing carbon dioxide (CO 2 ) emissions from industrial fermentation is an important challenge for sustainable bioprocess development.Saccharomyces cerevisiae, a major platform organism for ethanol production, emits substantial amounts of CO 2 during fermentation.In this study, heterologous CO 2 -fixation pathway-related genes were introduced into the xylose-fermenting S. cerevisiae strain DY2 to identify effective gene combinations for CO 2 -fixation.Eight recombinant strains were constructed using different combinations of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), phosphoribulokinase (PRK), and chaperone genes, including both conventional and unconventional combinations.Their anaerobic fermentation phenotypes were then evaluated.Improved ethanol production was observed only in strains expressing the form-I RuBisCO genes cbbL1, cbbS1 together with the PRK gene cfxP1, indicating that pathway performance depended largely on the compatibility between RuBisCO and PRK.The cbbLS/cfxP1/tHSP60-10 strain was selected as the final strain and designated DY2-LPH (cbbL1, cbbS1-cfxP1-tHSP60-10).Compared with the parental DY2 strain, this strain showed improved ethanol production under xylose and xylose-rich mixed-sugar conditions, whereas no clear advantage was observed under glucose-rich conditions.These results suggest that xylose metabolism provides a favorable background for the introduced RuBisCO-based CO 2 -fixation pathway and provide a basis for further development of CO 2 -fixing yeast strains.