Host-dependent activities of prenyltransferases in yeast and tobacco for cell-free cannflavin biosynthesis.

Abstract

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.