N,N-dimethyltryptamine (DMT) is a bioactive indole alkaloid that could greatly benefit from scalable, fermentation-based production for research and pharmaceutical applications. In this study, we reconstructed a two-step bacterial pathway converting L-tryptophan to DMT via tryptamine. This involved combining a pyridoxal 5'-phosphate (PLP)-dependent tryptophan decarboxylase from the bacterium Ruminococcus gnavus (RgnTDC) with an S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase from the cane toad Rhinella marina (RmNMT) in Escherichia coli. We optimised conditions for each step, determining 37 °C (pH 8.0) as the optimal condition for tryptamine production and 25 °C (pH 7.5) for DMT. While PLP supplementation did not raise tryptamine levels, methionine supplementation increased DMT levels by 2.8 times, emphasising the importance of methyl-donor supply. Co-culture and co-expression experiments showed that DMT accumulation depends on sufficient methylation capacity. Increased tryptophan availability led to tryptamine accumulation without a proportional increase in DMT formation, indicating a downstream limitation after decarboxylation. Together with the stimulatory effect of methionine supplementation, this result points to N-methylation and methyl-donor supply as key constraints in this system. In shake-flask cultures, a co-expression strain (TN1) produced 103 mg/L DMT after 48 h in complex medium without direct tryptophan supplementation. To enable growth in a defined medium, we used a workflow involving a tryptophan-enriched supernatant from a Corynebacterium glutamicum tryptophan overproducer, which supported de novo DMT formation at 16 mg/L in defined medium. These findings establish a plasmid-based platform for DMT production with E. coli and identify methyltransferase capacity as a key target for further yield improvements.
