Engineering yeasts as platform organisms for cannabinoid biosynthesis

- Expression of a functional active bacterial aromatic prenyltransferase in yeasts.
- Prenylation of olivetolic acid with geranyl diphosphate resulting in CBGA and 2-O-GOA.
- Functional expression of NphB and THCAS simultaneously.
- Reconstitution of the final biosynthetic cannabinoid pathway of C. sativa in yeast.

Δ9-tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa l. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme's coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.

Δ9-tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain, decreasing intraocular pressure in glaucoma or reducing spasticity associated with multiple sclerosis. Here we reconstituted the late biosynthetic pathway for THCA production from C. sativa in yeasts by combining the bacterial prenyltransferase NphB with the THCA forming enzyme. These engineered yeasts mark a significant step Highlightst &z.rtrif; oward the total biosynthesis of valuable cannabinoids in microbial hosts.126