Kutsyaba V. Interaction of regulatory genes for riboflavin biosynthesis and construction of riboflavin-overproducing strains in the yeast Pichia guilliermondii.

Object - regulation of riboflavin (RF) biosynthesis and iron acquisition in the yeast Pichia guilliermondii. The aim of the study - the investigation of the properties of regulatory mutants, the interaction of regulatory genes for RF biosynthesis and the influence of mutations leading to cell resistance to purine analogues on RF production in the yeast P. guilliermondii. Methods - biochemical, classical and molecular genetics, statistical. There was demonstrated that mutations rib80-22, rib83-6 are recessive, nucleous and monogenic. RIB83 and RIB80 genes participate in the regulation of both RF synthesis and iron acquisition in P. guilliermondii. There was shown that the genes RIB80 and RIB81, RIB81 and HIT1 manifested the synergic type of interaction between each other. Mutants with the combined mutations of rib83-6 with rib80-22, rib81-131 and rib80-22 rib81-131 were unable to overproduce RF and derepress synthesis the enzymes of flavinogenesis. The system for cloning of the positive regulatory geneRIB83 by means of functional complementation method using the triple mutant rib1-86 rib81 rib83 was designed. The strains carrying mutations rib80-22, rib81-131 and resistance to purine analogues 8-azaguanine and 4-aminopyrazolo[3,4-d]pyrimidine were obtained. They produced RF up to 1.7 g/L in synthetic medium when grown aerobically in laboratory fermenter. The addition of fodder RF obtained by means of the selected strain to a forage of hens had a positive effect on both body and egg weight. Area of use - metabolic engineering, biotehnology.