Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases
Metabolic Engineering June 14, 2026 Zachary N. Abrahms, Mohammad Majdi, Siena M. Madsen et al.
A 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.