(596e) Leveraging Enzyme Promiscuity to Enable Production of Non-Natural Psilocybin and Dmt Derivatives in E. coli

Our lab has published on the in vivo production of psilocybin in E. coli, which leverages the promiscuity of E. coli’s native tryptophan synthase b-subunit, TrpB, to synthesize the non-natural tryptophan derivative, 4-hydroxytryptophan, from serine and 4-hydroxindole. Here, we comprehensively investigate the substrate promiscuity of TrpB as well as the remainder of the enzymes in the psilocybin and N,N-dimethyltryptamine (DMT) pathways for the production of non-natural psilocybin and tryptamine derivatives. In order to facilitate the testing of various substituted indoles we expanded the biosynthesis pathway from previous work to include the cytochrome P450 monooxygenase, PsiH, and its cytochrome P450 reductase partner, CPR. We tested a panel of 50 substituted indoles in vivo to produce non-natural tryptophan and tryptamine derivatives using our modified E. coli strains. Substituted indoles that were successfully converted to tryptamine derivatives were then screened using the full psilocybin biosynthesis pathway spread across a two-strain co-culture system. This resulted in the production of 13 non-natural psilocybin derivatives, most representing the first example of production from a biological system. Additionally, two of the substituted indoles that processed into substituted tryptamines were used to produce substituted DMT derivatives by leveraging the human INMT enzyme to sequentially methylate the tryptamines. Ongoing work to evaluate the substrate promiscuity of engineered tryptophan synthases as well as diverse decarboxylases to further enhance the biosynthetic capabilities of our system will also be presented. This work serves to expand upon the production modalities for non-natural psilocybin and tryptamine derivatives that may have potential for therapeutic use.