Development of an E. coli-based norbaeocystin production platform and evaluation of behavioral effects in rats

Abstract

Interest in the potential therapeutic efficacy of psilocybin and other psychedelic compounds has escalated significantly in recent years. To date, little is known regarding the biological activity of the psilocybin pathway intermediate, norbaeocystin, due to limitations around sourcing the phosphorylated tryptamine metabolite for in vivo testing. To address this limitation, we first developed a novel E. coli platform for the rapid and scalable production of gram-scale amounts of norbaeocystin. Through this process we compare the genetic and fermentation optimization strategies to that of a similarly constructed and previously reported psilocybin producing strain, uncovering the need for reoptimization and balancing upon even minor genetic modifications to the production host. We then perform in vivo measurements of head twitch response to both biosynthesized psilocybin and norbaeocystin using both a cell broth and water vehicle in Long-Evans rats. The data show a dose response to psilocybin while norbaeocystin does not elicit any pharmacological response, suggesting that norbaeocystin and its metabolites may not have a strong affinity for the serotonin 2A receptor. The findings presented here provide a mechanism to source norbaeocystin for future studies to evaluate its disease efficacy in animal models, both individually and in combination with psilocybin, and support the safety of cell broth as a drug delivery vehicle.

Keywords: Depression; Head twitch response; Long-Evans rat; Norbaeocystin; Psilocybin; Psychedelic medicine.

Fig. 1

(a) Overview of study methods. Recombinant E. coli were developed capable of high-level norbaeocystin production. Norbaeocystin production was optimized and scaled up in a benchtop bioreactor. Norbaeocystin concentration in cell broth was quantified using HPLC. A rat with a magnet affixed to its head was gavaged with psilocybin or norbaeocystin in either a cell broth or water vehicle. A magnetometer coil was used in order to record head twitches. Waveforms were then analyzed to determine the head twitch count. (b) Norbaeocystin biosynthesis pathway. The E. coli strain contains three genes, one native (trpB) and two heterologous (psiD, psiK) that enable norbaeocystin biosynthesis from external supplementation of 4-hydroxyindole. Tryptophan synthase (TrpB) condenses 4-hydroxyindole and serine to form 4-hydroxytryptophan. P. cubensis tryptophan decarboxylase (PsiD) converts 4-hydroxytryptophan into 4-hydroxytryptamine while releasing a carbon dioxide and water. Finally, P. cubensis kinase (PsiK) converts 4-hydroxytryptamine into norbaeocystin using a phosphate donated by ATP.

Fig. 2

Summary of genetic optimization and scale-up results. (a) Promoter library screening. Individual colonies from the operon (red bars) and pseudooperon (gray bars) libraries were selected and evaluated to discover elite production strains. Data for samples producing 0 mg/L of norbaeocystin (11.5% of total colonies screened) are not shown. (b) Normalized production of operon library members for norbaeocystin and psilocybin pathways organized in order of increasing promoter strength: G6 (low) – T7 (high). Constructs for each operon promoter configuration were identified and screened providing evidence that the transcriptional optimization solution for the norbaeocystin pathway differed from that of the psilocybin pathway. (c) Effect of varying supplemental serine concentration in the initial fermentation media on strain performance in the bioreactor. (d) Metabolite and growth curve profiles for a representative norbaeocystin bioreactor fed-batch fermentation. Data shown for one replicate of the 0 g/L serine condition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Dose-dependent effects of tryptamines alone and in combination when administered in a broth vehicle. (a) Psilocybin caused significant increases in the number of head twitches at 1.0 mg/kg. (b) Norbaeocystin did not increase the number of head twitches at any dose studied. Note: *p < 0.05; **p < 0.01.

Fig. 4

Dose-dependent effects of tryptamines alone and in combination when administered in a water vehicle. (a) Psilocybin caused significant increases in the number of head twitches at 0.2, 1.0, and 2.0 mg/kg. (b) Norbaeocystin did not increase the number of head twitches at any dose studied. Note: *p < 0.05; **p < 0.01.