Four enzymes from button bush enable de novo biosynthesis of spirooxindole alkaloids in yeast
Bibliographic record
Abstract
Oxindoles are a structurally unique subclass of monoterpenoid indole alkaloids (MIAs) with significant medicinal values such as neuroprotective and anticancer properties and are predominantly found in the Rubiaceae plant family. Recent research has only just begun to uncover the biosynthetic pathways of this complex MIA subclass. Here, we report the discovery, characterization, and functional reconstitution of a four-enzyme pathway from Cephalanthus occidentalis (button bush) that enables the complete de novo biosynthesis of heteroyohimbine-type oxindole alkaloids in engineered yeast. Through transcriptomic mining and biochemical validation, we identified and characterized an ajmalicine synthase (CoAJS), a heteroyohimbine/yohimbine/corynanthe C3-oxidase (CoHYC3O), a C3-reductase (CoHYC3R), and an oxindole synthase (CoOIS) capable of converting strictosidine aglycone into 3-epi-ajmalicine and subsequently into the oxindole alkaloids mitraphylline and isomitraphylline. Saturation kinetics of CoOIS revealed comparable catalytic efficiencies for 3-epi-ajmalicine and its C20 epimer akuammigine, indicating substrate tolerance within the heteroyohimbine subclass. Yeast-based reconstruction of the pathway successfully yielded both mitraphylline epimers and the spirooxindole uncarine F. This work elucidates the enzymatic logic behind oxindole formation and expands the toolkit for MIA scaffold diversification and synthetic biology.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".