Betaine-Driven Methyl Supply System to Improve Psilocybin Production in E. coli
2022 Annual Meeting
Betaine-Driven Methyl Supply System to Improve Psilocybin Production in E. coli
The Activated Methyl Cycle (AMC) is essential for the regeneration of S-Adenosyl-L-Methionine (SAM), which serves as the methyl donor for nearly all methyltransferase enzymes in nature. When introducing a methyltransferase-containing heterologous pathway into E. coli, a metabolic imbalance within the AMC can occur that results in the buildup of toxic metabolites resulting in reduced titer, rate, and yield. While direct methionine supplementation can serve as an AMC bypass to increase SAM availability, this approach is still hampered by the accumulation of toxic intermediates such as S-Adenosyl-Homocysteine (SAH) and homocysteine. Introduction of a betaine catabolic pathway in E. coli, catalyzed by betaine-homocysteine methyltransferase, BHMT, is an alternative route for methionine regeneration from homocysteine and enables a bypass of the native folate cycle used by MetE and MetH in the native AMC. This can serve as a route to enhance AMC activity without increasing metabolite pool size, decreasing concentration of toxic intermediate products.
Another enzyme with potential to augment the AMC, SAHase, would reduce the native Mtn and LuxS genes with a one-step process, which is hypothesized to decrease metabolic burden on the cell and subsequently increase production. Lastly, SAM2 will be investigated as a replacement for the native MetK enzyme which converts methionine to SAM. Here, we show the introduction of these three alternative AMC enzymes into E. coli metabolism and evaluate their potential to enhance the production of methylated products, namely the psychedelic drug candidate, psilocybin. To evaluate this system we screened 5 pathway variants across a series of transcriptionally varied conditions. The best performers were selected and will be tested in bench-top scale bioreactors and compared to our current best producing strains. This new auxiliary pathway has the potential to overcome the limitations of metabolic burden faced by current production strains leading to a more competitive and industrially relevant biosynthetic production process.
