Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation

Abstract

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P_TDH3, P_FBA1, and P_TDH1 were favorable for high expression, and P_SED1, P_HXT7, P_PDC1, P_TEF1, P_TPI1, and P_PGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P_TEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. P_ZWF1 and P_SOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P_ALD3 and P_TKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.

Keywords: Cultivation; Fermentation; Hemicellulose; Microarray; Promoter; Reporter gene assay; Saccharomyces cerevisiae; Xylose; Yeast.