Thirty years after the discovery of native xylose fermenting yeasts, metabolic engineering is creating strains that can handle industrial feedstocks under commercial conditions. A highly functional system for the transformation and genetic manipulation of Scheffersomyces (Pichia) stipitis is now available. The system is based on LoxP-flanked synthetic selectable markers (nat1 and hph) that confer resistance to nourseothricin and hygromycin during integration followed by excision with an engineered version of Cre on vectors carrying resistance to zeocin and bleomycin (Sh ble). These have been used to construct ca. 50 individual transformants of S. stipitis that carry various combinations of native and synthetic genes for sugar transport, xylose metabolism and fermentation. Our engineered strains produce 57 g/l ethanol in 49 h from pure sugars. Two sequenced strains of S. stipitis (CBS 6054 and CBS 5773) show significant differences at both physiological and genome levels. S. stipitis has a highly functional mating system for crossing engineered strains, and we have selected hybrids with significant resistance to hydrolysates. Spathaspora passalidarum, to which we are currently adapting our transformation technologies, will ferment xylose to ethanol faster than it ferments glucose and it will co-ferment cellobiose and xylose or glucose and xylose when the glucose level is below 30 g/l. Adapted strains produce 38 to 40 g/l ethanol from hydrolysate containing 2 to 2.4 g/l acetic acid. Fermentation kinetics/ transcriptomics/metabolomics studies with S. stipitis and S. passalidarum cultivated in bioreactors show that the latter yeast is well suited to oxygen limited growth on xylose.
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