Engineering cannabinoid production in Saccharomyces cerevisiae

Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. The production was significantly increased by introducing a fusion protein consisting of ERG20^WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56 mg L^-1 . The implementation of the cannabinoid synthase genes enabled the production of Δ⁹ -tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed-batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2 mg L^-1 and 117 mg L^-1 , respectively.