Thaxtomins are a group of cyclic diketopiperazines that are virulence factors of many plant- pathogenic Streptomyces strains which cause the common scab potato disease. Thaxtomins represent an economically valuable and agriculturally important natural herbicide because of their ability to inhibit cellulose synthesis. It is desirable to perfect a natural synthesis route because the process is more efficient, economical, and environmentally friendly than existing chemical synthesis routes. In addition, thaxtomin analogs may represent value in the medical field due to their anti- fungal, antiviral, and antibiotic properties. Our lab developed a cell- free biocombitorial approach to synthesizing thaxtomin analogs. Biosynthesis of 124 unique thaxtomin analogs was achieved in vitro directly from simple amino acid components. This was made possible through the novel functional characterization of three biosynthetic thaxtomin enzymes: TxtA, TxtB, and TxtC. The production and functions of these enzymes was explored with the previously characterized TxtE enzyme. Furthermore, our lab demonstrated rare P450 cytochrome enzymatic activity that is capable of catalyzing both aliphatic and aromatic hydroxylation in one process. Both hydroxylation processes are valuable to biosynthesis reactions, specifically, they allowed for the production of over 40 additional novel diketopiperazine products.