Chapter 2 Recent Advances in Construction of the Efficient Producers of Riboflavin and Flavin Nucleotides (FMN, FAD) in the Yeast Candida famata Dariya V. Fedorovych, Kostyantyn V. Dmytruk, and Andriy A. Sibirny Abstract 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 flavino- genic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohy- drolase 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. Key words Riboflavin, Flavin mononucleotide, Flavin adenine dinucleotide, Yeast Candida famata, Metabolic engineering, Riboflavin, FMN and FAD overproducers 1 Introduction Riboflavin (vitamin B 2 ) is one of the most important vitamins required for human and animal nutrition. This vitamin is metabolic precursor of flavin nucleotides involved as coenzymes in numerous Maria Barile (ed.), Flavins and Flavoproteins: Methods and Protocols, Methods in Molecular Biology, vol. 2280, https://doi.org/10.1007/978-1-0716-1286-6_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021 15