Improved production of the non-native cofactor F₄₂₀ in Escherichia coli

The deazaflavin cofactor F₄₂₀ is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F₄₂₀ biosynthesis to optimize F₄₂₀ production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F₄₂₀ biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space-time yield of F₄₂₀ compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F₄₂₀-production system and will allow the recombinant in vivo use of F₄₂₀-dependent enzymes for biocatalysis and protein engineering applications.