Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases.
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 pathwa
- Authors
- Abrahms ZN, Majdi M, Madsen SM, Morton CJ, Sen AK, Fried NB, Sawyer LE, Cegielski ER, Spezzano SJ, Jones JA
- Journal
- Metabolic engineering
- Published
- 2026-09-01
- DOI
- 10.1016/j.ymben.2026.102490
- PMID
- 42288133
- Content type
- Paper
- Authority
- Academic
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.
Citation
Abrahms ZN, Majdi M, Madsen SM, Morton CJ, Sen AK, Fried NB, Sawyer LE, Cegielski ER, Spezzano SJ, Jones JA (2026) Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases.. Metabolic engineering. doi:10.1016/j.ymben.2026.102490
DOI: 10.1016/j.ymben.2026.102490
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