Metabolic Engineering of <i>Escherichia coli</i> for Efficient Production of Redox Cofactors Flavin Mononucleotide and Flavin Adenine Dinucleotide

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential redox cofactors in cellular metabolism. In this study, riboflavin-producing <i>Escherichia coli</i> was engineered for de novo FMN and FAD synthesis. The RibF_M-1 mutant with defective FAD synthase activity was overexpressed to produce FMN. NADH supply was enhanced by overexpressing <i>sthA</i> and deleting <i>pntAB</i>, while deletion of <i>ndh</i> encoding NADH dehydrogenase II and <i>appB</i> encoding cytochrome oxidase bd-II improved ATP regeneration. The resulting strain FMN08 produced 3457.3 mg/L FMN in fed-batch fermentation. FMNAT from <i>Candida glabrata</i> was engineered by semirational design to increase catalytic activity 2.14-fold, and coexpression of FMN1 from <i>Saccharomyces cerevisiae</i> enabled FMN-to-FAD conversion. Deletion of <i>ushA</i> encoding 5'-nucleotidase and overexpression of <i>purA</i> further promoted FAD production. The final strain FAD13 produced 2305.2 mg/L FAD in fed-batch fermentation. These results represent the highest reported titers of de novo synthesis of these redox cofactors.