Xylitol production from wheat straw by genetically engineered yeast

Xylitol, a natural sugar alcohol, is widely used as sugar substitute in various foods and pharmaceuticals. In recent years, industrial interest in xylitol has been steadily increasing. It has comparable sweetening power, albeit 40 % less calories and an about 10 times lower glycaemic index than conventional sucrose. It has been demonstrated that Candida guilliermondii, a non-conventional yeast, naturally produces xylitol and therefore offers a biotechnological alternative to the current chemical method of xylitol production. The aim of our study was to produce increased amounts of xylitol by fermentation of pre-treated wheat straw with genetically engineered C. guilliermondii strains. First, we optimized pre-treatment strategies of wheat straw in order to enable appropriate growth of C. guilliermondii. In detail, we amended the procedure in such a way that increased the amounts of fermentable sugars and reduced the amounts of growth inhibitory substances generated by the pre-treatment processes. Moreover, we deciphered molecular players responsible for generation of xylitol in this yeast. We identified two enzymes involved in the xylose metabolic pathway that play a crucial role in xylitol production: xylose reductase (XYL1) and xylose dehydrogenase (XDH1). By molecular cloning, we generated an XYL1 overexpressing strain, an XDH1 knockout strain, and strains that combined both modifications. We confirmed the successful genetic alterations in xylose metabolism by genetic analysis and enzyme activity tests. Finally, we investigated the effect of the single and combined genetic modifications on xylitol yield and used the best-performing strain for bio-catalytic conversion of wheat straw-based sugars into xylitol.