Yatsyshyn V. Metabolic engineering of the nonconventional yeast Candida famata aimed at constructing overproducers of flavin nucleotides

Object of research: flavin nucleotides biosynthesis and regulation of this process in yeast. The aim of the work: development of approaches to construction of FMN and FAD overproducers of flavinogenic yeasts Candida famata, using the metabolic engineering methods. Methods: microbiological, biochemical, molecular genetic, mathematical statistics. The Debaryomyces hansenii FMN1 and FAD1 genes were cloned. The scheme of construction of the flavin nucleotides overproducers allowed obtaining C. famata recombinant strains with increased FMN and FAD production. Replacing of the native promoter of D. hansenii FMN1 gene on a strong promoter TEF1 C. famata led to 200-fold increasing of RF-kinase activity and 1000-fold increasing of FMN content in the culture of C. famata recombinant strains. The recombinant strains of C. famata, which contain the FAD1 gene driven by the TEF1 promoter demonstrated 7-15-fold increase in FAD synthetase activity and accumulated 450 mg/l FAD.The medium components significant for FMN and FAD production were identified. The optimization of cultivation medium led to a 29-36-fold increase in the FMN production and 28,5-fold increase in the FAD production. Area of use: applied microbiology, biotechnology, biochemistry.