Host-dependent activities of prenyltransferases in yeast and tobacco for cell-free cannflavin biosynthesis
- 1Plant Biomaterials and Biotechnology Division, Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju-si, 54874 Republic of Korea
- 2Department of Systems Biology, Institute of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 Republic of Korea
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.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13068-026-02800-3.
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BibTeX
@article{lee2026hostdependent,
title = {{Host-dependent activities of prenyltransferases in yeast and tobacco for cell-free cannflavin biosynthesis}},
author = {Sung-eun Lee and Saet Buyl Lee and Hyun Ju Jung and Ji-Su Kim and Hyoseon Choi and Sangchul Choi and Beom-Gi Kim},
journal = {Biotechnology for Biofuels and Bioproducts},
year = {2026},
month = jul,
doi = {10.1186/s13068-026-02800-3},
url = {https://doi.org/10.1186/s13068-026-02800-3},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Host-dependent activities of prenyltransferases in yeast and tobacco for cell-free cannflavin biosynthesis
AU - Sung-eun Lee
AU - Saet Buyl Lee
AU - Hyun Ju Jung
AU - Ji-Su Kim
AU - Hyoseon Choi
AU - Sangchul Choi
AU - Beom-Gi Kim
PY - 2026
DA - 2026/07/29
JO - Biotechnology for Biofuels and Bioproducts
DO - 10.1186/s13068-026-02800-3
UR - https://doi.org/10.1186/s13068-026-02800-3
AB - 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. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13068-026-02800-3.
SN - 2731-3654
ER -