{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T21:08:50Z","timestamp":1778360930144,"version":"3.51.4"},"posted":{"date-parts":[[2026,5,5]]},"group-title":"Synthetic Biology","reference-count":46,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T00:00:00Z","timestamp":1777939200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01AT012633"],"award-info":[{"award-number":["R01AT012633"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["MCB-2338009"],"award-info":[{"award-number":["MCB-2338009"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["DBI-2019674"],"award-info":[{"award-number":["DBI-2019674"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["IOS-2220733"],"award-info":[{"award-number":["IOS-2220733"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Plant monoterpene indole alkaloids (MIAs) exhibit important pharmacological activities, yet understanding of their biosyntheses remains incomplete. Since protein-protein interactions (PPIs) represent a conserved regulatory mechanism in MIA-producing plants, we developed a large-scale, yeast-based screening pipeline to profile PPIs of a key enzyme, strictosidine \u03b2-D-glucosidase (SGD) from\n                  <jats:italic>Mitragyna speciosa<\/jats:italic>\n                  (kratom). This screen identified six novel medium-chain dehydrogenases\/reductases (MDRs) as high-confidence interaction partners of SGD. Biochemical characterization revealed that all six MsMDRs produce an MIA we named charlamine by acting directly on the reactive strictosidine aglycone intermediate, preventing its spontaneous rearrangement and establishing a functional rationale for SGD\u2013MDR interaction. One MsMDR additionally catalyzed the reduction of vallesiachotamine, derived from the spontaneous rearrangement of strictosidine aglycone, to another previously unreported MIA, vallesiachotaminol. Parallel transcriptomics and genomics analyses uncovered a biosynthetic gene cluster containing a dihydrocorynantheine aldehyde esterase, functioning downstream of MsMDRs. Collectively, these findings demonstrate the utility of interactomics-driven plant pathway discovery.\n                <\/jats:p>\n                <jats:p>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"722234v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:p>","DOI":"10.64898\/2026.05.01.722234","type":"posted-content","created":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T04:35:28Z","timestamp":1778042128000},"source":"Crossref","is-referenced-by-count":0,"title":["Interactomics-driven discovery of alkaloid biosynthetic pathways in kratom"],"prefix":"10.64898","author":[{"given":"Bo","family":"Yang","sequence":"first","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samuel","family":"Kemiji","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"},{"name":"Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianing","family":"Han","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"},{"name":"Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, 08544, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dong Oh","family":"Han","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yinan","family":"Wu","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chang","family":"Liu","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frank C.","family":"Schroeder","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA"},{"name":"Boyce Thompson Institute, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0364-5632","authenticated-orcid":false,"given":"Sijin","family":"Li","sequence":"additional","affiliation":[{"name":"Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026050913200425000_2026.05.01.722234v1.1","doi-asserted-by":"publisher","DOI":"10.1039\/b512615k"},{"key":"2026050913200425000_2026.05.01.722234v1.2","doi-asserted-by":"crossref","first-page":"1551","DOI":"10.1038\/s41589-023-01430-2","article-title":"Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast","volume":"19","year":"2023","journal-title":"Nature Chemical Biology"},{"key":"2026050913200425000_2026.05.01.722234v1.3","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1007\/s11101-015-9406-4","article-title":"Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites","volume":"15","year":"2016","journal-title":"Phytochemistry reviews"},{"key":"2026050913200425000_2026.05.01.722234v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.1203877"},{"key":"2026050913200425000_2026.05.01.722234v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.pbi.2022.102200"},{"key":"2026050913200425000_2026.05.01.722234v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2015.02.006"},{"key":"2026050913200425000_2026.05.01.722234v1.7","doi-asserted-by":"crossref","first-page":"12116","DOI":"10.1038\/ncomms12116","article-title":"Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity","volume":"7","year":"2016","journal-title":"Nature Communications"},{"key":"2026050913200425000_2026.05.01.722234v1.8","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1038\/s41467-017-00154-x","article-title":"A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate","volume":"8","year":"2017","journal-title":"Nature Communications"},{"key":"2026050913200425000_2026.05.01.722234v1.9","doi-asserted-by":"crossref","first-page":"e202307995","DOI":"10.1002\/ange.202307995","article-title":"Discovering dynamic plant enzyme complexes in yeast for kratom alkaloid pathway identification","volume":"135","year":"2023","journal-title":"Angewandte Chemie"},{"key":"2026050913200425000_2026.05.01.722234v1.10","doi-asserted-by":"crossref","first-page":"23","DOI":"10.2147\/SAR.S164261","article-title":"Current perspectives on the impact of Kratom use","volume":"10","year":"2019","journal-title":"Substance Abuse and Rehabilitation"},{"key":"2026050913200425000_2026.05.01.722234v1.11","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1007\/s11101-017-9509-1","article-title":"How to prove the existence of metabolons?","volume":"17","year":"2018","journal-title":"Phytochemistry Reviews"},{"key":"2026050913200425000_2026.05.01.722234v1.12","doi-asserted-by":"crossref","unstructured":"Carr, S.C. & O\u2019Connor, S.E. A Tight-Knit Family: The Medium-Chain Dehydrogenase\/Reductases of Monoterpene Indole Alkaloid Biosynthesis. Biochemistry (2025).","DOI":"10.1021\/acs.biochem.5c00234"},{"key":"2026050913200425000_2026.05.01.722234v1.13","doi-asserted-by":"publisher","DOI":"10.1021\/acs.orglett.1c00206"},{"key":"2026050913200425000_2026.05.01.722234v1.14","doi-asserted-by":"crossref","unstructured":"Brose, J. et al. The Mitragyna speciosa (Kratom) Genome: a resource for data-mining potent pharmaceuticals that impact human health. G3 Genes|Genomes|Genetics 11(2021).","DOI":"10.1093\/g3journal\/jkab058"},{"key":"2026050913200425000_2026.05.01.722234v1.15","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1721469115"},{"key":"2026050913200425000_2026.05.01.722234v1.16","doi-asserted-by":"crossref","first-page":"2274","DOI":"10.1021\/jo00245a027","article-title":"Synthesis of vallesiachotamine","volume":"53","year":"1988","journal-title":"The Journal of Organic Chemistry"},{"key":"2026050913200425000_2026.05.01.722234v1.17","doi-asserted-by":"crossref","first-page":"2177","DOI":"10.3390\/plants14142177","article-title":"LC-HRMS Coupling to Feature-Based Molecular Networking to Efficiently Annotate Monoterpene Indole Alkaloids of Alstonia scholaris","volume":"14","year":"2025","journal-title":"Plants"},{"key":"2026050913200425000_2026.05.01.722234v1.18","doi-asserted-by":"crossref","first-page":"1934578X1801300508","DOI":"10.1177\/1934578X1801300508","article-title":"eliciting effect of catharanthine on the biosynthesis of vallesiachotamine and isovallesiachotamine in catharanthus roseus Cambial Meristematic Cells","volume":"13","year":"2018","journal-title":"Natural Product Communications"},{"key":"2026050913200425000_2026.05.01.722234v1.19","first-page":"118","article-title":"Density functional theory approach towards bioactivity analysis of Isovallesiachotamine natural bio molecule","volume":"7","year":"2019","journal-title":"Int J Sci Res Phys Appl Sci"},{"key":"2026050913200425000_2026.05.01.722234v1.20","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.3390\/biology11101492","article-title":"A Chromosome-Scale Genome Assembly of Mitragyna speciosa (Kratom) and the Assessment of Its Genetic Diversity in Thailand","volume":"11","year":"2022","journal-title":"Biology"},{"key":"2026050913200425000_2026.05.01.722234v1.21","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1021\/acs.jnatprod.3c00092","article-title":"Metabolite and Molecular Characterization of Mitragyna speciosa Identifies Developmental and Genotypic Effects on Monoterpene Indole and Oxindole Alkaloid Composition","volume":"86","year":"2023","journal-title":"Journal of Natural Products"},{"key":"2026050913200425000_2026.05.01.722234v1.22","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2024.09.011"},{"key":"2026050913200425000_2026.05.01.722234v1.23","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.3390\/biology11101492","article-title":"A Chromosome-Scale Genome Assembly of Mitragyna speciosa (Kratom) and the Assessment of Its Genetic Diversity in Thailand","volume":"11","year":"2022","journal-title":"Biology"},{"key":"2026050913200425000_2026.05.01.722234v1.24","doi-asserted-by":"publisher","DOI":"10.1111\/nph.19162"},{"key":"2026050913200425000_2026.05.01.722234v1.25","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c13644"},{"key":"2026050913200425000_2026.05.01.722234v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/msb.2010.53"},{"key":"2026050913200425000_2026.05.01.722234v1.27","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201200328"},{"key":"2026050913200425000_2026.05.01.722234v1.28","doi-asserted-by":"publisher","DOI":"10.1186\/1746-4811-8-25"},{"key":"2026050913200425000_2026.05.01.722234v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2010.08.007"},{"key":"2026050913200425000_2026.05.01.722234v1.30","doi-asserted-by":"crossref","first-page":"9628","DOI":"10.3390\/ijms15069628","article-title":"A cautionary note on the use of split-YFP\/BiFC in plant protein-protein interaction studies","volume":"15","year":"2014","journal-title":"International journal of molecular sciences"},{"key":"2026050913200425000_2026.05.01.722234v1.31","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.62337"},{"key":"2026050913200425000_2026.05.01.722234v1.32","doi-asserted-by":"publisher","DOI":"10.1039\/D2NP00032F"},{"key":"2026050913200425000_2026.05.01.722234v1.33","doi-asserted-by":"crossref","first-page":"25233","DOI":"10.1021\/jacs.5c02863","article-title":"Enzymatic Basis of Stereochemical Control in Reserpine Biosynthesis","volume":"147","year":"2025","journal-title":"Journal of the American Chemical Society"},{"key":"2026050913200425000_2026.05.01.722234v1.34","doi-asserted-by":"crossref","first-page":"5620","DOI":"10.1016\/j.cell.2024.08.027","article-title":"A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes","volume":"187","year":"2024","journal-title":"Cell"},{"key":"2026050913200425000_2026.05.01.722234v1.35","doi-asserted-by":"crossref","unstructured":"Zhang, Y. & Fernie, A.R. Metabolons, enzyme\u2013enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism. Plant Communications 2 (2021).","DOI":"10.1016\/j.xplc.2020.100081"},{"key":"2026050913200425000_2026.05.01.722234v1.36","doi-asserted-by":"crossref","unstructured":"Besseau, S. et al. Evolution of CYP71D drives the diversification of monoterpene indole alkaloid biosynthesis in Gentianales. Plant Communications (2025).","DOI":"10.1101\/2025.01.09.632084"},{"key":"2026050913200425000_2026.05.01.722234v1.37","doi-asserted-by":"crossref","first-page":"e202210934","DOI":"10.1002\/ange.202210934","article-title":"Expansion of the catalytic repertoire of alcohol dehydrogenases in plant metabolism","volume":"134","year":"2022","journal-title":"Angewandte Chemie"},{"key":"2026050913200425000_2026.05.01.722234v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.09.011"},{"key":"2026050913200425000_2026.05.01.722234v1.39","doi-asserted-by":"crossref","first-page":"3030","DOI":"10.1021\/acschembio.0c00706","article-title":"An untargeted approach for revealing electrophilic metabolites","volume":"15","year":"2020","journal-title":"ACS chemical biology"},{"key":"2026050913200425000_2026.05.01.722234v1.40","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1038\/s41467-022-28391-9","article-title":"Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C. elegans","volume":"13","year":"2022","journal-title":"Nature communications"},{"key":"2026050913200425000_2026.05.01.722234v1.41","doi-asserted-by":"publisher","DOI":"10.1021\/ac051437y"},{"key":"2026050913200425000_2026.05.01.722234v1.42","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1038\/s41467-026-68881-8","article-title":"Discover the maze-like network for glabridin biosynthesis","volume":"17","year":"2026","journal-title":"Nature Communications"},{"key":"2026050913200425000_2026.05.01.722234v1.43","doi-asserted-by":"crossref","unstructured":"Stander, E.A. et al. The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis. G3 Genes|Genomes|Genetics 12 (2022).","DOI":"10.1093\/g3journal\/jkac268"},{"key":"2026050913200425000_2026.05.01.722234v1.44","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1038\/s42003-023-05574-8","article-title":"The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids","volume":"6","year":"2023","journal-title":"Communications Biology"},{"key":"2026050913200425000_2026.05.01.722234v1.45","article-title":"Gggenomes: effective and versatile visualizations for comparative genomics","year":"2024","journal-title":"arXiv preprint"},{"key":"2026050913200425000_2026.05.01.722234v1.46","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.01.722234","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T20:20:17Z","timestamp":1778358017000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.01.722234"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,5]]},"references-count":46,"URL":"https:\/\/doi.org\/10.64898\/2026.05.01.722234","relation":{},"subject":[],"published":{"date-parts":[[2026,5,5]]},"subtype":"preprint"}}