Work

This work is a result of meaningful research and is devoted to construction of microorganisms with high production of biofuels, such as fuel ethanol and butanol. Lignocellulosic biomass offers a high potential as a feedstock for alternative energy sources. The main compounds of lignocellulose are glucose and C5-sugar xylose. One of the bottlenecks in the pathway from lignocellulosic residues to fuel ethanol is the absence of the microbial strains capable to efficiently convert xylose to ethanol. Therefore, the development of industrial yeast strains with high productivity of xylose alcoholic fermentation is an important task. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved, this goal can be achieved applying metabolic engineering approaches. The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (up to 50 °C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation (SSF). However, the efficiency of xylose conversion to ethanol in H. polymorpha is low and not sufficient for industrial production. As a result of the conducted studies a number of limiting steps in xylose conversion to ethanol in H. polymorpha were found. Applying metabolic engineering approaches and classical selection we were able to obtain H. polymorpha recombinant strains with ability to synthesize 0.3 g of ethanol per 1g of consumed xylose at 45 °С. The modified form of the xylose reductase of Pichia stipitis with decreased affinity toward NADPН was created via site-specific mutagenesis. Recombinant strain of P. stipitis overexpressing the modified xylose reductase characterized by 1,3-fold increase in productivity of xylose alcoholic fermentation was constructed. The rates of alcoholic fermentation of hydrolyzates of wheat bran and sawdust were also improved by 1,5 times as compared to the wild-type strain. The aim of this work was also to obtain new efficient domestic producers of butanol. The microbiological analysis of sludge and soil of water in Kiev was conducted for search of new strains, resulting in isolation of bacteria similar to Clostridium. As a result three prospective strains were identified as C. tyrobutyricum, C. acetobutyicum and C. butyricum. The first one was characterized by production of butyric acid and other two strains were producers of butanol. The scientific works series consists of 37 publications, including 11 papers (7 of them are published in the international journals) and 26 abstracts in the collected materials of the home and international congresses and conferences.