Dmytruk O. Metabolic engineering of the initial stages of xylose catabolism in the yeast Hansenula polymorpha aimed at constructing improved producers of ethanol

Object of research: the first steps of xylose metabolism in thermotolerant yeast H. polymorpha. The aim of work: construction of recombinant strains of thermotolerant yeasts H. polymorpha capable to produce an increased amount of ethanol during xylose fermentation by the way of modification of the first steps of xylose metabolism. Methods of research: microbiological, molecular biological, genetic, biochemical, statistical. For the first time, xylA genes encoding xylose isomerase from E. coli and S. сoelicolor were successfully overexpressed in yeast. The recombinant strain of H. polymorpha, which simultaneously overexpressed E. coli xylA and H. polymorpha XYL3 was constructed, the ethanol production of xylose by this strain increased 3,5-4 times compared with the wild type. Site-specific mutagenesis of the H. polymorpha XYL1 gene was carried out which decreased affinity of this enzyme toward NADPН(H+) for 17 times compared with the native enzyme. H. polymorpha strain overexpressing the mutated XYL1 gene together with native xylitol dehydrogenase and xylulokinase was constructed. The ethanol production from xylose by this strain increased nearly 7,4 times and the xylitol production decreased 5 times compared with the wild type strain. Area of use: applied microbiology, biotechnology, biochemistry.