Voronovs'kyj A. Development of transformation systems for flavinogenous yeasts Pichia guilliermondii and Candida famata

The dissertation contains results of the work on development of host-vector transformation systems for flavinogenous yeasts Pichia guilliermondii and Candida famata. A number of P. guilliermondii and C. famata auxotrophic mutants were tested for complementation by corresponding selectable marker genes. P. guilliermondii strains RG21 (hisx rib1), RG130 (hisx rib7), RG162 (hisx rib7), and C. famata R7 (rib1), R8 (rib7), L203 (leu2), L2012 (leu2), L20105 (leu2), 8-33 (leu2 rib7) deficient in GTP cyclohydrolase (rib1) or riboflavin synthase (rib7), or beta-isopropylmalate dehydrogenase (leu2) were selected as recipients for the host-vector transformation systems. P. guilliermondii RIB1 and RIB7 genes and the Saccharomyces cerevisiae LEU2 gene were selected and served as markers for these systems. The P. guilliermondii ARS (designated PgARS) was discovered and localised in the 3' flanking region of the RIB1 gene. Five C. famata DNA fragments carrying ARSs were cloned. Escherichia coli-flavinogenous yeasts shuttle vectors were constructed. The C. famata genomic library was created based on this yeast's replicative vector. The gene encoding riboflavin synthase (RIB7) was cloned from this library by complementation of rib7 mutation in the strain 8-33. It was shown that cloned C. famata RIB7 gene is expressed in the P. guilliermondii.