(246a) De Novo Production of the Naturally Occurring Hallucinogen, 5-Meo-Dmt, in E. coli

O-methyltransferases (OMTs) play a crucial role in the biosynthesis of natural products ranging from small molecules to lignin. These methyltransferases require a methyl donor, such as SAM, to regioselectively methylate hydroxyl groups on substrates. We explore the promiscuity of 12 OMTs from 5 plant species, M. tuberculosis, and humans to realise their potential in bridging previously reported heterologous metabolic pathways in E. coli. Here, we report the de novo production of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) from glucose through the coupling of 5-hydroxytryptophan biosynthesis catalysed by an engineered phenylalanine 4-hydroxylase (P4H) from X. campestris which is sequentially decarboxylated to serotonin by Psilocybe cubensis PsiD, O-methylated by an OMT characterized in this work, and N-methylated by human indolethylamine N-methyltransferase (INMT) to complete the pathway. Our lab has previously demonstrated both decarboxylation and N-methylation of tryptamine which we combine with OMT and P4H modules to produce a route to 5-MeO-DMT de novo. We further balance metabolic flux in this pathway by generating a library of over 400 pathway variants using multiple characterized promoter strengths and pathway architectures.