Pilot Scale Production of a F₄₂₀ Precursor Under Microaerobic Conditions

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. F_O 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 F_O from an engineered Escherichia coli host strain. We were able to shift the cofactor synthesis from riboflavin to F_O via the implementation of oxygen-limited process conditions during heterologous fbiC expression and reached a final titer of 5.05 mg L^-1 F_O 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 F_O formation and confirmed growth-associated F_O biosynthesis.