Xylose fermentation by Saccharomyces cerevisiae using endogenous xylose-assimilating genes.

OBJECTIVES: To genetically engineer Saccharomyces cerevisiae for improved ethanol productivity from glucose/xylose mixtures. RESULTS: An endogenous gene cassette composed of aldose reductase (GRE3), sorbitol dehydrogenase (SOR1) and xylulose kinase (XKS1) with a PGK1 promoter and a terminator was introduced into two S. cerevisiae strains, a laboratory strain (CEN.PK2-1C) and an industrial strain (Kyokai No. 7). The engineered Kyokai No. 7 strain (K7-XYL) exhibited a higher sugar consumption rate (1.03 g l(-1) h(-1)) and ethanol yield (63.8 %) from a glucose and xylose mixture compared to the engineered CEN.PK2-1C strain. Furthermore, K7-XYL produced a larger amount of ethanol (39.6 g l(-1)) compared to K7-SsXYL (32 g l(-1)) with integrated xylose reductase and xylitol dehydrogenase from a xylose-assimilating yeast Scheffersomyces stipitis instead of GRE3 and SOR1. CONCLUSION: The created S. cerevisiae strain showed sufficient xylose-fermenting ability to be used for efficient ethanol production from glucose/xylose.

Kinetic modeling and sensitivity analysis for higher ethanol production in self-cloning xylose-using Saccharomyces cerevisiae.

We constructed a xylose-utilizing Saccharomyces cerevisiae strain using endogenous xylose-assimilating genes (strain K7-XYL). Such self-cloning yeast is expected to make a great contribution to cost reduction of ethanol production processes. However, it is difficult to modify self-cloning yeast for optimal performance because the available gene source is limited. To improve the ethanol productivity of our self-cloning yeast, a kinetic model of ethanol production was constructed and sensitivity analysis was performed. Alcohol dehydrogenase (ADH1) was identified as a metabolic bottleneck reaction in the ethanol production pathway. An ADH1 overexpression strain (K7-XYL-ADH1) was constructed and evaluated in YP (yeast extract 10 g/L, peptone 20 g/L) medium containing 50 g/L xylose as the sole carbon source. Strain K7-XYL-ADH1 showed higher ethanol productivity (13.8 g/L) than strain K7-XYL (12.5 g/L). Then, K7-XYL-ADH1 was evaluated in YP medium containing 80 g/L glucose and 50 g/L xylose; however, the ethanol productivity did not change relative to that of K7-XYL (K7-XYL 46.3 g/L, K7-XYL-ADH1 45.9 g/L). We presumed that due to the presence of glucose, the internal redox balance of the cells had changed. On culturing in an aerated 5-L jar fermentor to change the internal redox balance of cells, strain K7-XYL-ADH1 showed higher ethanol productivity than K7-XYL (K7-XYL 45.0 g/L, K7-XYL-ADH1 49.4 g/L). Our results confirmed that ADH1 was a metabolic bottleneck in the ethanol production pathway. By eliminating the bottleneck, self-cloning yeast showed almost the same ethanol productivity as genetically modified yeast.