Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Abstract

Abstract Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH2PO4, glycine, and (NH4)2SO4. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.