Optimization of Δ9-tetrahydrocannabinolic acid synthase production in Komagataella phaffii via post-translational bottleneck identification.

Δ⁹–Tetrahydrocannabinolic acid (THCA) is a secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain or reducing spasticity associated with multiple sclerosis. Here, we investigated the influence of the co-expression of 12 helper protein genes from Komagataella phaffii (formerly Pichia pastoris) on the functional expression of the Δ⁹–tetrahydrocannabinolic acid synthase (THCAS) heterologously expressed in K. phaffii by screening 21 clones of each transformation. Our findings substantiate the necessity of a suitable screening system when interfering with the secretory network of K. phaffii. We found that co-production of the chaperones CNE1p and Kar2p, the foldase PDI1p, the UPR-activator Hac1p as well as the FAD synthetase FAD1p enhanced THCAS activity levels within the K. phaffii cells. The strongest influence showed co-expression of Hac1s – increasing the volumetric THCAS activities 4.1-fold on average. We also combined co-production of Hac1p with the other beneficial helper proteins to further enhance THCAS activity levels. An optimized strain overexpressing Hac1s, FAD1 and CNE1 was isolated that showed 20-fold increased volumetric, intracellular THCAS activity compared to the starting strain. We used this strain for a whole cell bioconversion of cannabigerolic acid (CBGA) to THCA. After 8 h of incubation at 37 °C, the cells produced 3.05 g L^-1 THCA corresponding to 12.5% g_THCA g_CDW^-1.