The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the above mentioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2 mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5 mg/L was achieved.
Keywords: FMN and FAD overproducers; Flavin adenine dinucleotide; Flavin mononucleotide; Metabolic engineering; Riboflavin; Yeast Candida famata.