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Metabolic engineering for construction of efficient yeast strains producing biologically active substances and biofuels

Work number - M 29 AWARDED

Authors: Kurylenko O.O., Lyzak O.O., Semkiv M.V., Yatsyshyn V.Yu.

Presented by the Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine

In present series of the work known and original metabolic engineering approaches were successfully applied for construction of yeast producers of biologically active substances ‒ riboflavin (RF), flavin nucleotides (FMN and FAD), glutathione, glycerol and biofuel (ethanol).

The Candida famata stable overproducer of vitamin B2 was obtained by amplification of SEF1 gene encoding positive regulator of RF synthesis, as well as increased expression of the RIB1 and RIB7 genes encoding key enzymes of RF biosynthesis. The optimal cultivation conditions for maximum RF production (over 10 g/l in the laboratory conditions) were selected. The recombinant strains of the yeast C. famata overproducing flavokinase (200-fold increase as compared to the wild type strain), FMN (385 mg/l in the optimized medium) and FAD (450 mg/l in the optimized medium) were obtained by replacing of the native promoter of the FMN1 (encodes flavokinase) and/or FAD1 (encodes FAD synthetase) genes of the yeast C. famata with a strong constitutive promoter TEF1.

Recombinant Hansenula polymorpha strain overexpressing both GSH2 gene, encoding γ-glutamylcysteine synthetase, and MET4 gene, coding for transcription activator of the genes involved in cysteine biosynthesis (precursor of glutathione) was obtained applying metabolic engineering approaches. Conditions for efficient glutathione production by recombinant H. polymorpha strain were optimized and the semi-industrial model for glutathione production was developed. With the aim of enhancing glycerol production in Saccharomyces cerevisiae strain with decreased triose phosphate isomerase activity, increased glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase activities and expression of modified form of membrane transporter Fps1 was constructed. Described strain produced 8 times more glycerol than wild type strain.

For improvement of ethanol production in yeast S. cerevisiae strains with decreased intracellular ATP level were constructed. For instance, strain with derepressed unspecific alkaline phosphatase, that can disrupt ATP, produced up to 13% more ethanol than initial strain. Applying metabolic engineering approaches and classical selection we were able to obtain Hansenula  polymorpha recombinant strains with ability to synthesize 0.3 g of ethanol per 1g of consumed xylose at 45 °С. Recombinant strain of Pichia stipitis overexpressing the modified  xylose reductase characterized by 1,3-fold increase in ethanol productivity during xylose alcoholic fermentation. The parameters 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 results of studies on the subject are set out in 11 chapters in monograph, 22 articles (including 12 in foreign journals), 6 Ukraine Patents, 2 International Patents and 114 abstracts. The total number of bibliographic references (according to Google Scholar database – 85, h-index of authors is 1-4. Total number of publications of the authors is 155.