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Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to(More)
Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for d-xylose utilization through the heterologous expression of genes for aldose reductase (XYL1), xylitol dehydrogenase (XYL2), and d-xylulokinase (XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing(More)
The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first(More)
Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on d-xylose. When a gene for d-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered(More)
Several proteins with significant identity to ubiquitin have been characterized recently. In contrast to ubiquitin's main role in targeting proteins for degradation, a described function of one family of ubiquitin-related proteins, the Rub family, is to serve as a stable post-translational modification of a complex involved in the G1-to-S cell cycle(More)
D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S. cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism(More)
Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces(More)
In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1-Cdc53-F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for(More)
We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single(More)
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