Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5'-phosphate) can functionally substitute the natural deazaflavin F420 and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae. Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F420 yield of native F420-producing organisms such as Mycobacterium smegmatis, but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L-1 h-1 and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae, in vivo FOP production by SpRFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a SpRFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP.