High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains.

Abstract

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30\u202f°C and 40\u202f°C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40\u202f°C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40\u202f°C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402\u202fg/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.