Using enzymes to speed up chemical reactions can help make those reactions more environmentally friendly. Some enzymes need small helper molecules, called cofactors, in order to function properly. F420 is one such cofactor. Because it readily donates electrons to other molecules, especially those that do not typically react with other cofactors, F420 could be a more sustainable alternative to chemical processes that have no biological substitute yet. One difficulty of using F420 is that it either needs to be extracted from slow growing microorganisms such as Mycobacterium smegmatis, or needs to be synthesized chemically, both producing only a small amounts. We therefore designed an artificial cofactor, named FO-5’-phosphate (FOP), which mimics the chemical properties of F420 but can be produced much more efficiently. The first step in creating FOP is to chemically assemble its precursor (FO), which can then be converted to FOP using enzymes. In the first study, we used protein engineering to design a mutant enzyme that was capable of carrying out this last step in the process. In the second study, we designed a way to manufacture FOP inside of fast growing E. coli cells, by introducing enzymes from other organisms in this bacterium, and showed that this method was even more efficient than using Mycobacterium smegmatis to produce F420. Lastly, and most importantly, we found that enzymes that are dependent on F420 work just as well with FOP as they do with F420, which indicates that FOP can be a viable and scalable alternative.