Objective: To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome.
Methods: Based on S. cerevisiae CEN. PK 113-5D, we constructed xylose-metabolizing strains where the promoter of xylulokinase gene XKS1 was replaced by TEF1 promoter, PGK1 promoter and HXK2 promoter on the chromosome. We quantitated the transcriptional level of XKS1 gene (accumulated mRNA) and measured the activity of xylulokinase in each stains. Furthermore, we also determined the intracellular level of ATP and evaluated the xylose-fermenting abilities of the engineered strains.
Results: The engineered strains exhibited higher expression of xylulokinase than the parental strain at both transcription and enzyme activity levels. The highest xylulokinase activity was observed in the strain whose XKS1 was controlled by PGKlp, and was decreasingly followed by the strains whose XKS1 was controlled by TEF1p, HXK2p and native promoter. The expression level of xylulokinase negatively correlated with intracellular level of ATP and positively correlated with ability of ethanol production from xylose. The highest ethanol yield was 0.35 g/g consumed sugars while the lowest xylitol yield, which was 0.18 g/g consumed xylose, was observed.
Conclusion: By promoter replacement, xylulokinase was overexpressed at different levels. In this work, higher expressional level of xylulokinase improved the conversion of xylose to ethanol.