Development and Application of T7 Promoter Libraries to Enable Psilocybin Production in Vibrio Natriegens

Title: Development and Application of T7 Promoters Libraries to enable Psilocybin Production in Vibrio natriegens

Author List:

Claire M. Cashdollar

John D. Brinton

Alexandra M. Adams

J. Andrew Jones

Psilocybin is a psychedelic prodrug that occurs naturally in “magic mushrooms”, which include over 200 species of fungi, most notably the Psilocybe cubensis. Psilocybin is becoming increasingly important in the medical field, as this drug has been shown to be an effective treatment for a multitude of mental disorders, as well as addiction. Previously, commercial production of psilocybin has been achieved through heterologous gene expression in the highly engineerable E. coli.

Vibrio natriegens is a gram-negative bacterium that has recently been highlighted as a desirable host for protein synthesis; they have the fastest growth rate known for a non-pathogenic microbe. There are many potential applications for V. natriegens in bioengineering, including as a host for the for the recombinant production of psilocybin.

Here, we study the gene expression of the psiD, psiK, and psiM genes, with the purpose of producing psilocybin and its intermediates through a batch-fed in vivo approach utilizing a 4-hydroxyindole starting substrate. Because of the rapid-growing nature of V. natriegens, these organisms are very appealing for commercial use. Their fast growth enables robust processes with enhanced productivity leading to promising industrial processes with short residence times and limited opportunity for contamination due to the rapid growth.

Here, we implement the first instance of a T7 promoter optimization strategy in Vibrio natriegens. Promoter libraries were constructed for V. natriegens using the ePathOptimize approach and evaluated in E. coli and V. natriegens. Interestingly, we found that although the native T7 sequence demonstrated high strength in both hosts, the reduced strength promoters showed a large degree of host specificity. Three T7-variant promoters (T7 consensus, E2, and F2) were developed and used to construct three promoter libraries in operon, pseudooperon, and monocistronic forms. The psiD, psiK, and psiM genes were cloned into each of these promoter library configurations and were screened for norbaeocystin and psilocybin. Efforts are ongoing to develop enhanced production methods for psilocybin in V. natriegens, however, preliminary results and comparison to an E. coli host indicate that V. natriegens may not be the most suitable host for this pathway.