A truly enabling new approach by @taralowensohn.bsky.social Will Cody and @sattelylab.bsky.social to probe plant genetics at scale.

Single-gene-per-cell delivery coupled to an effective transcriptional selection system

www.biorxiv.org/content/10.1...

Also crazy that the long-term-storage genome can be ~20x larger

And it’s chopped up into the just the useful pieces to form the get-shit-done genome (doi.org/10.1371/jour...)

Almost like ciliates have encrypted their genome 🤯

Thank you to @frankelab.bsky.social for his eloquent overview in Nature news on our Taxol paper.

He deftly articulates the history of this longstanding biochemistry challenge.

rdcu.be/eqwOy

We also found a side chain gene that enabled us to push the pathway beyond baccatin III and synthesize, de novo, debenzoyl-deoxy-taxol, a molecule 2 steps removed from Taxol. The 8 genes we found, with @skampranis.bsky.social's recent work on these last 2 steps, now complete the pathway to Taxol

These discoveries let us engineer a 17-gene pathway into tobacco to produce baccatin III at levels comparable to the concentrations in yew. Because baccatin the current industrial precursor extracted from yew plants to produce Taxol, this represents a major step towards its sustainable production

A second major big was that transient modifications (absent from Taxol) appear necessary for some intermediate enzymes. 5 acetylations are needed, while baccatin/Taxol have only 3. Co-expression analysis led us to both the acetyltransferase to install these and the de-acetylases to remove them.

Beyond FoTO1, our approach identified enzymes that fill in the gaps for Taxol biosynthesis. By combining our new discoveries with previously known enzymes, we built up the pathway towards Taxol

Among them was FoTO1, from a family with no precedent in plant metabolism. FoTO1 completely resolves the dramatic inefficiencies of the first oxidation reaction, where T5aH produces mostly side products (blue). With FoTO1, it produces only the correct product (red). Mechanistic research ongoing!

We applied our new approach approach to find the missing Taxol synthesis genes in the yew genome. We found that known Taxol genes cluster into 3 coexpressed modules, not one. Within these modules, we found 8 new genes in the Taxol pathway, including several biological and chemical surprises.

Thanks to heroic previous work, we know some of the enzymes in the Taxol pathway. But 1) we were missing many enzymes and 2) all reconstitution efforts go off the rails at the second step. That oxidation, by T5aH, mostly makes incorrect products, suggesting we were missing something

Yew has thousands of specialized metabolism genes but how do they fit into individual pathways? To differentially activate pathways, we devised a scaled strategy for tissues perturbation. With snRNA-seq we capture of 10^5 cells across 100s of samples. Importantly, this works with non-model organisms

Co-expression (genes with correlated activation) is our main tool for identifying plant gene sets, but often lacks the resolution needed in the huge plant genomes. For Taxol, we need ~20 genes from the yew genome (3x larger than ours, barely annotated). Find 20 needles in a 50000-gene haystack

Taxol shows how hard it is to find valuable plant genes. This yew-derived blockbuster cancer drug (billions/yr for 25yr) has treated millions, but we still harvest from plants (4 trees per patient!) because it's too complex to synthesize affordably & we hadn’t found a complete biosynthetic gene set