(160ah) Metabolic Engineering Techniques to Enable and Increase De Novo Production of Psilocybin in E. coli

As proof-of-principle metabolic engineering projects are pushed toward commercialization, significant roadblocks caused by the introduction of metabolically demanding heterologous pathways must be overcome. These roadblocks include low product yields, reduced growth rates, and genetic instability among others. Many of these problems can be attributed to perturbations to the complex, highly evolved regulatory networks found in natural anabolic pathways in E. coli. Here we present the development and evaluation of a tailored E. coli platform strain constructed to improve production of high-value chemicals from tryptophan or indole precursors, with de novo synthesis of the hallucinogenic tryptamine, psilocybin, as a goal. Key precursor (serine, tryptophan) and cofactor (ATP, SAM) regeneration pathways were targeted through random optimization screening of a priori selected metabolic targets. Methods include regulatory gene knockouts, genomic promoter engineering, and other gene silencing techniques to achieve an industrially viable strain, capable of enhanced psilocybin biosynthesis, both de novo and from a 4-hydroxyindole substrate. The modular and stepwise approach to strain optimization described in this work is generalizable to a wide range of high-value, small molecule target products, and thus, will be of wide interest to the field.