(160ag) In Vivo Biosynthesis of Dimethyltryptamine (DMT) in E. coli

N,N-Dimethyltryptamine (DMT), a tryptamine derivative found naturally in wide range of plants and animals, including humans, is classified as a psychoactive chemical. Clinical trials involving the administration of ayahuasca, a concoction containing DMT, has been shown to help treat mental disorders such as anxiety and depression. DMT containing beverages (e.g., ayahuasca) have been used in religious contexts in South America for centuries. Only recently has modern medicine become interested in formally investigating the healing properties of DMT in a clinical setting. This pharmaceutical interest motivates the need for robust and efficient manufacturing strategies, as isolation of DMT from natural sources is quite difficult due to DMT’s low and variable natural abundance coupled with limited reserves of plant matter with sufficiently high concentrations for extraction. Advances in both metabolic and genetic engineering, make it possible to engineer recombinant microbes to produce DMT to meet growing demand for ongoing pre-clinical and clinical studies to evaluate the safety and efficacy of DMT. Here, we present the development of a biosynthetic production pathway for DMT in the model microbe Escherichia coli. Through the application of genetic optimization techniques and the use of benchtop bioreactors operated under pH-stat conditions, the in vivo production of DMT was observed for the first time in E. coli. Through further transcriptional and fermentation optimization of our lead strain, we were able to observe milligram-level de novo production of DMT from glucose. Despite low initial titers, we continue to improve production using classic metabolic engineering approaches focused on enhancing precursor supply and cofactor regeneration coupled with deletion of competing pathways and deregulation of complex regulatory systems in amino acid biosynthesis.