Evaluation of Decarboxylase Promiscuity and Suitability for Diverse Tryptamine Biosynthesis

Psilocybin, the key psychoactive molecule in magic mushrooms, has demonstrated psychotherapeutic properties in preclinical and clinical studies. The biosynthesis of psilocybin has been previously established using three enzymes of interest: PsiD, PsiK, and PsiM. PsiD is a decarboxylase responsible for converting tryptophan to tryptamine. PsiD is known to be a promiscuous enzyme, in fact, to enable psilocybin biosynthesis in E. coli it efficiently catalyzes the decarboxylation of the non-natural substrate, 4-hydroxytryptophan; however, it fails to act on bulkier and more reactive substrates. It is crucial to identify more promiscuous pathway enzymes as we push to enable the biosynthesis of a larger drug candidate pool. In this work, we evaluated the enzymes PsmH, RgnTDC, and CroTDC. PsmH is an enzyme within the biosynthetic gene cluster that produces physostigmine. PsmH natively acts on 5-hydroxytryptophan, suggesting it naturally has evolved a larger substrate binding pocket. CroTDC (Catharanthus roseus L-tryptophan decarboxylase) has been shown to effectively decarboxylate tryptophan in S. cerevisiae, more commonly known as brewer’s yeast. RgnTDC (Ruminococcus gnavus L-tryptophan decarboxylase) has been shown to have approximately one hundred and thirty times more catalytic efficiency in comparison to its counterpart CroTDC. Here we evaluate the substrate specificity of these engineered decarboxylases, the impact of expression strength on their functional activity, and the ability of these enzymes to expand the synthesis of psilocybin derivatives in an E. coli host.