Kava (Piper methysticum) is an ethnomedicinal shrub native to the Polynesian islands with well-established anxiolytic and analgesic properties. Its main psychoactive principles, kavalactones, form a unique class of polyketides that interact with the human central nervous system through mechanisms distinct from those of conventional psychiatric drugs. However, an unknown biosynthetic machinery and difficulty in chemical synthesis hinder the therapeutic use of kavalactones. In addition, kava also produces flavokavains, which are chalconoids with anticancer properties structurally related to kavalactones. Here, we report de novo elucidation of the key enzymes of the kavalactone and flavokavain biosynthetic network. We present the structural basis for the evolutionary development of a pair of paralogous styrylpyrone synthases that establish the kavalactone scaffold and the catalytic mechanism of a regio- and stereo-specific kavalactone reductase that produces a subset of chiral kavalactones. We further demonstrate the feasibility of engineering styrylpyrone production in heterologous hosts, thus opening a way to develop kavalactone-based non-addictive psychiatric therapeutics through synthetic biology.
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The sequences of the genes reported in this article have been deposited in NCBI GenBank (accessions MK058492–MK058514). Protein expression plasmids are available from Addgene (see Supplementary Table 5). The raw sequencing reads have been submitted to NCBI SRA (accession PRJNA494686) and the de novo assembled transcriptome to NCBI TSA (accession GHAC00000000). Raw metabolomic LC–MS datasets have been uploaded to the GNPS-MassIVE database (accessions MSV000083272 and MSV000083274–MSV000083277). Protein structures have been deposited in Protein Data Bank (accessions 6OP5, 6CQB and 6NBR).
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This work was supported by grants from the Smith Family Foundation, Edward N. and Della L. Thome Memorial Foundation, the Family Larsson-Rosenquist Foundation and the National Science Foundation (grant no. CHE-1709616). T.P. is a Simons Foundation Fellow of the Helen Hay Whitney Foundation. J.-K.W is supported by the Beckman Young Investigator Program, Pew Scholars Program in the Biomedical Sciences (grant no. 27345) and the Searle Scholars Program (grant no. 15-SSP-162). This work is on the basis of research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on NE-CAT 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. RNA-seq service was provided free of charge by the Beijing Genome Institute in exchange for an evaluation of their BGISEQ-500 sequencing platform. We thank B. Marotta for an introduction to kava, C. Nguyen and F. A. Samatey for advice regarding crystallography, G. Fink for providing yeast strains and expression vectors and Weng lab members for constructive comments.
T.P. and J.-K.W. have filed a patent application on metabolic engineering of kavalactones and flavokavains using the enzymes discovered in this study. J.-K.W. is a co-founder, a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, which develops biotechnologies related to natural products. All other authors have no competing interests.
Peer review information: Nature Plants thanks Fernando Geu-Flores and Reuben Peters and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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