Cannflavins are prenylated flavones found in Cannabis sativa that exhibit potent anti-inflammatory activities, but their low natural abundance limits pharmacological exploitation. Biotechnological strategies are therefore needed to support cannflavin production. Here, we evaluated yeast ( Saccharomyces cerevisiae ) and Nicotiana benthamiana as heterologous platforms for cell-free cannflavin biosynthesis. Six plant- and microbe-derived prenyltransferases (PTs) were expressed in both hosts and assessed for apparent cannflavin-forming activity using cell-free enzyme assays. In yeast-derived microsomal assays, CsPT3 from C. sativa produced cannflavin A (CFL-A) and cannflavin B (CFL-B), and removal of its predicted N-terminal transit peptide enhanced apparent CFL-A and CFL-B formation. By contrast, CsPT3 and cytCsPT3 did not produce detectable cannflavins in N. benthamiana -derived crude extract assays. Instead, the soluble microbial PT NphB supported CFL-A formation in N. benthamiana -derived enzyme preparations. We further established a crude extract-based one-pot assay in N. benthamiana and examined the effects of codon optimization of NphB, assay pH, and Agrobacterium inoculum density on apparent CFL-A formation. These results demonstrate that cannflavin-forming PT activity is highly dependent on host context and expression conditions. This comparative cell-free approach identifies suitable enzyme–host combinations for cannflavin biosynthesis and provides a practical platform for screening and improving prenylated flavonoid production.
Lee et al. (Wed,) studied this question.