GENETICALLY-ENGINEERED SACCCHAROMYCES YEAST CAPABLE OF EFFECTIVE COFERMENTATION OF GLUCOSE AND XYLOSE

Xylose is one of the major fermentable sugars present in cellulosic bi omass, second only to glucose. However, Saccharomyces spp., the best s ugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. i nto xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2 mu m-based high-copy-number yeast-a. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resis tance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylul okinase gene (from Saccharomyces cerevisiae). These xylose-metabolizin g genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. stra in 1400 with each of these plasmids resulted in the conversion of stra in 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectiv ely utilizes xylose for aerobic growth. Furthermore, the resulting rec ombinant yeasts also have additional extraordinary properties. For exa mple, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presenc e of xylose for induction, nor is the synthesis repressed by the prese nce of glucose in the medium. These properties make the recombinant ye asts able to efficiently ferment xylose to ethanol and also able to ef ficiently coferment glucose and xylose present in the same medium to e thanol simultaneously.