Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases
Zachary N. Abrahms, Mohammad Majdi, Siena M. Madsen, Chloe J. Morton, Abhishek K. Sen, Niya B. Fried, Lily E. Sawyer, Evelyn R. Cegielski, Sean J. Spezzano, J. Andrew Jones
Metabolic Engineering June 14, 2026 DOI: 10.1016/j.ymben.2026.102490 via OpenAlex
Summary
AI-generated from the abstractA new genome engineering strategy called ePathIntegrate uses CRISPR-associated transposases to stably insert complex metabolic pathways into the chromosome of E. coli. When plasmid-optimized pathways for the psychedelic compounds psilocybin and DMT were moved directly to the genome, productivity dropped because promoters behaved differently in the new context. A library of mutant T7 promoters was developed to restore proper transcriptional control. With ePathIntegrate, the re-optimized pathways yielded 1.88 g/L psilocybin and 1.62 g/L DMT in fed-batch bioreactors. Whole-genome sequencing showed precise on-target integration but also some off-target integrations and small mutations, indicating both the promise and current limitations of this approach.
Study at a glance
| Characteristics | Experimental study Peer reviewed |
|---|---|
| Population | Escherichia coli |
| Interventions | ePathIntegrate CRISPR-associated transposases mutant T7 promoters |
| Topics | Psilocybin |
| Keywords | Transposase Escherichia coli Biosynthesis Dna transposable elements |
| Key finding | ePathIntegrate enabled genome-encoded E. coli strains that produce 1.88 g/L psilocybin and 1.62 g/L DMT, but revealed off-target integrations and mutations that limit the approach. |
Abstract
Stable, high-level biosynthesis of complex natural products requires precise control of heterologous pathway expression, yet transcriptional architectures optimized on plasmids often fail when transferred to the chromosome. Here, we present ePathIntegrate, a genome-centric pathway engineering strategy that leverages CRISPR-associated transposases (CASTs) to integrate and rebalance multigene metabolic pathways in Escherichia coli. Direct genomic transfer of plasmid-optimized psilocybin and N,N-dimethyltryptamine (DMT) pathways resulted in a loss of productivity, driven by context-dependent promoter behavior. To address this, we developed and characterized a library of mutant T7 promoters that restore mid-range transcriptional control on the genome. Applying ePathIntegrate enabled re-optimization of both pathways, yielding genome-encoded strains that achieve 1.88 g/L psilocybin and 1.62 g/L DMT in fed-batch bioreactors. Whole-genome sequencing of CAST-mediated strains further revealed (i) precise on-target integration, (ii) some off-target pathway integrations, and (iii) small mutations in a subset of strains, highlighting both the power and limitations of CAST-mediated strain engineering.