Characterization of Catechol O-Methyltransferase Activity and Promiscuity in E. coli

Heterologous enzyme expression in E. coli often experiences nuanced activity and substrate preferences. Our aim was to study 14 catechol O-methyltransferases (COMT) individually and in combination with a tryptophan decarboxylase (PsiD) to determine the preferred metabolic route towards the synthesis of the naturally occurring hallucinogen, 5-MeO-DMT. An intermediate in the pathway, 5-hydroxytryptophan (5-HTP), can either be decarboxylated first by PsiD and methylated second by COMT (Route 1), or methylated first and decarboxylated second - both routes leading to the production of 5-MeO-tryptamine. To determine the preferred metabolic route in E. coli BL21star^TM(DE3), we cloned a 4 member mutant T7 promoter library for each of the 14 COMTs individually and in combination with PsiD (polycistronic configuration). Individual colonies of BL21 star^TM(DE3) expressing the promoter library (xx⁴) were grown in a semi-defined medium (AMM) and supplemented with pertinent intermediates. The fermentation-based screening was carried out in 48-well plates over 48h at 37ºC. The samples were centrifuged and supernatants were analyzed by HPLC-MS. The data suggests that COMTs prefer 5-HTP over serotonin as a substrate while PsiD is able to decarboxylate 5-HTP and 5-MeO-tryptophan equally well. Pairing this information in the context of the biosynthetic routes available leads to a clearly preferred metabolic route which will guide the use of COMTs and PsiD in the development of extended pathways towards various tryptamine natural products.