(593d) Development of Design Rules Guiding the Transition of Biosynthetic Pathways from Plasmid to Genome – a Psilocybin Biosynthesis Case Study

Due to the ease of multiplexed pathway assembly, abundance of characterized synthetic biology parts, and simplicity of strain construction, most heterologous microbial biosynthesis pathways are initially developed and optimized on plasmids, rather than the genome. However, prior to commercialization, a stable genome integrated biosynthetic pathway is desired for bioprocess development at scale. This transition from plasmid to genome is rather unpredictable due to a dearth of available data, and frequently poses a technical hurdle slowing the commercialization timeline until reoptimization efforts yield acceptable and competitive production metrics.

Here, we present the development of an E. coli-based, genome-integrated, psilocybin biosynthetic platform. Psilocybin is the psychoactive chemical that is responsible for the vivid hallucinogenic episodes experienced upon ingestion of ‘magic’ mushrooms and represents a promising drug candidate for the treatment of many mental health disorders. We will highlight the lessons learned in the transition from a plasmid-based production process to a genomic-based pathway expression system. We will present mCherry-based functional characterization of seven engineered promoters, expressed from six genomic locations, as compared with plasmid-based expression from identical constructs. In general, expression from the genome resulted in lower mCherry signal when compared to plasmid, however some constructs were exceptions to this general trend. Additionally, we will explore the impact of biosynthetic pathway performance by comparing: (1) a single operon at a single genomic location, (2) split pathway expression across multiple genomic locations, and (3) multi-copy expression from the genome. After optimization, the genome integrated psilocybin production strain resulted in significantly higher psilocybin titers as compared with the original, optimized plasmid-based strain. Final characterization of this elite strain with regards to pathway stability and general robustness to process variables will be discussed.