D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeast can ferment xylose and xylulose more than twice as fast as the parent yeast.
A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.
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