The functional investigation of redox cofactors is important for many potential biocatalytic processes, yet limited access to these molecules is often hampering efficient research activities. Deazaflavin-dependent enzymes mediate a range of biochemical redox reactions in prokaryotes. Coenzyme F420-dependent enzymes are interesting for asymmetric redox biocatalysis and other challenging reactions, but low F420 titers harvested from natural producers and engineered host strains so far limit intensive investigation of these enzymes. FO is a natural precursor of F420, which already shares many of the redox properties and was previously confirmed as a surrogate for F420 in certain enzymes. Here, we focused our research on the development of an overall process workflow from a 30-L pilot scale stirred tank bioprocess to an optimized downstream purification process to obtain pure FO from an engineered Escherichia coli host strain. We were able to shift the cofactor synthesis from riboflavin to FO via the implementation of oxygen-limited process conditions during heterologous fbiC expression and reached a final titer of 5.05 mg L-1 FO in our fermentation broth, which for the first time allows the purification of relevant amounts for subsequent experiments. Online fluorescence measurement within the bioreactor system made it possible to monitor FO formation and confirmed growth-associated FO biosynthesis.