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 Peer reviewed DOI: 10.1016/j.ymben.2026.102490 via OpenAlex
Summary
The study introduces ePathIntegrate, a method for engineering metabolic pathways in Escherichia coli using CRISPR-associated transposases. This strategy was applied to optimize the biosynthesis of psilocybin and N,N-dimethyltryptamine (DMT), achieving yields of 1.88 g/L and 1.62 g/L respectively in bioreactors. While the method successfully restored transcriptional control, some off-target integrations and mutations were also observed, indicating both its effectiveness and limitations.
Study at a glance
| Population | Escherichia coli strains |
|---|---|
| Key finding | ePathIntegrate enabled re-optimization of psilocybin and DMT pathways, yielding genome-encoded strains that produce 1.88 g/L psilocybin and 1.62 g/L DMT. |
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.