Phytocannabinoids, a group of specialized metabolites that accumulate in the glandular trichomes of Cannabis sativa L., are compounds of high economic value recognized for both their recreational and medicinal purposes. Cannabinoids exert their effects in the body by binding to endocannabinoid receptors localized to the central and peripheral nervous system, and components of the immune system. Modulation of these systems by cannabinoids has shown efficacy in a myriad of therapies, including treatment of pain management, epilepsy, asthma, inflammation and neurodegenerative disease. Cannabinoids are produced primarily in the flowering trichomes of cannabis plants through the enzymatic convergence of a resorcinol core (featuring an alkyl side chain) and a monoterpene moiety derived from geranylpyrophosphate (GPP). The biosynthetic pathways responsible for the production of both of these compounds have largely been determined in the past few decades, allowing for partial reconstruction in heterologous system. While these systems show promise for the sourcing of cannabinoids, improvements are necessary for economical production on a larger scale. Low yields of cannabinoids in these systems suggest that there may be unidentified accessory proteins that assist in the biosynthesis of these compounds. Our current understanding suggests that cannabinoid biosynthesis is primarily limited by the production of OA from malonyl-CoA and hexanoyl-CoA. This research project aimed to create synthetic biology system(s) that incorporate the enzymatic components for the OA-forming steps of the cannabinoid biosynthetic pathway into microbial hosts, S. cerevisiae and E. coli. to facilitate the investigation of a potential chalcone isomerase-like (CHI-L) accessory protein in said pathway. Accessory proteins such as CHI-L’s are known to interact with synthases and to be necessary for the processing of the polyketide intermediate, and observed function of similar CHI-L proteins in other embryophytes suggest a mechanism of action for this proposed CsCHI-L in cannabis. While the experimentation was ultimately successful in creating synthetic biology systems capable of incorporating relevant enzymes and producing the intermediate compound OA, there was no observable difference when comparing compound production in systems with the proposed CsCHI-L protein and those without. This data is indicative that the CsCHI-L protein does not function in these early steps of the pathway.