Abstract
Meyerozyma guilliermondii, a non-conventional yeast that naturally assimilates xylose, is considered as a candidate for biotechnological production of the sugar alternative xylitol. Because the genes of the xylose metabolism were yet unknown, all efforts published so far to increase the xylitol yield of this yeast are limited to fermentation optimization. Hence, this study aimed to genetically engineer this organism for the first time with the objective to increase xylitol production. Therefore, the previously uncharacterized genes of M. guilliermondii ATCC 6260 encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) were identified by pathway investigations and sequence similarity analysis. Cloning and overexpression of the putative XR as well as knockout of the putative XDH genes generated strains with about threefold increased xylitol yield. Strains that combined both genetic modifications displayed fivefold increase in overall xylitol yield. Enzymatic activity assays with lysates of XR overexpressing and XDH knockout strains underlined the presumed functions of the respective genes. Furthermore, growth evaluation of the engineered strains on xylose as sole carbon source provides insights into xylose metabolism and its utilization for cell growth.
Highlights
The interest on the five-carbon sugar alcohol xylitol has been constantly rising over the last years
KU141F1 and all strains derived thereof are originated from the reference strain M. guilliermondii ATCC 6260 (American Type Culture Collection, ATCC Manassas, USA)
Activity EC 1.1.1.9 (d-xylulose reductase or NAD + -dependent xylitol dehydrogenase), which catalyzes the reaction from d-xylulose to xylitol and vice versa, was linked to PGUG_05726 of the M. guilliermondii ATCC6260 genomic scaffold 8
Summary
The interest on the five-carbon sugar alcohol xylitol has been constantly rising over the last years. This is because it is metabolized in an insulin independent manner and, is suitable for people suffering from diabetes, and because of the rising interest in lowcalorie diets (Ylikahri 1979). Xylitol tastes as sweet as sucrose whilst harboring about 40% less calories (Zacharis 2012). Its anti-cariogenic properties make xylitol an interesting agent for the pharmaceutical industry (Mäkinen 1979). Xylitol is produced chemically, which has some disadvantages in terms of energy demand, wastewater pollution and extensive purification requirements. The need of pure d-xylose as raw material for a nickelcatalyzed hydrogenation is one of the main cost rising factors, as this d-xylose has to be purified from mainly
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