Han, D. O. | Search bioRxiv

bioRxiv2026

Interactomics-driven discovery of alkaloid biosynthetic pathways in kratom

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 {beta}-D-glucosidase (SGD) from Mitragyna speciosa (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-MDR 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/722234v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ccc22dorg.highwire.dtl.DTLVardef@1e03f3borg.highwire.dtl.DTLVardef@15773deorg.highwire.dtl.DTLVardef@782ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Identification and Optimization of Kratom Strictosidine Pathway Enabled by Yeast Multiplex Engineering

Monoterpene indole alkaloids (MIAs) are a major class of plant natural products with important pharmaceutical activities, yet the biosynthetic pathway to their universal precursor, strictosidine, has been fully elucidated in only Catharanthus roseus. In kratom (Mitragyna speciosa), only the first and last steps of strictosidine biosynthesis were previously known. Here, we applied multiplex pathway engineering in yeast to accelerate the discovery, reconstruction, and optimization of the kratom strictosidine pathway. Iterative multiplex integration and screening identified 13 functional kratom genes and enabled rapid validation of functional pathway modules, thereby completing the kratom strictosidine pathway from geranyl pyrophosphate and tryptophan. We also identified a vacuolar secologanin transporter, MsNPF2.6, which increased strictosidine production by 62% in yeast. Pathway optimization through the incorporation of nepetalactol-producing enzymes from other plants further supported strictosidine production in yeast from fed geraniol and tryptophan. These results establish the strictosidine pathway in kratom and highlight multiplex engineering as a powerful platform for rapid plant pathway discovery and optimization.

bioengineering↗