Cofactor F420 is a low-redox potential enzyme cofactor that mediates a number of otherwise recalcitrant reactions in the central metabolism of methanogens and in the secondary metabolism of Actinobacteria. The low redox potential (−340 mV cf. −320 mV for NAD and −220 mV for flavins) and unusual activity of F420 make it attractive for use in biocatalysis, however its narrow taxonomic distribution in organisms that are difficult to grow and for which synthetic biology tools are underdeveloped, has limited investigation into its potential applications. We have recently proposed a revised biosynthetic pathway for F420 and demonstrated its function in E. coli with modest yields. Here we build upon that work through developing a genome-scale metabolic model for F420 production in E. coli. F420 concentration was increased from 0.41 to 0.86 µmol g−1 DCW by overexpressing PEP synthase with glucose as carbon source. Growth on succinate increased concentrations up to 2 µmol g−1 however with compromised growth and low biomass.