Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast

Abstract

Heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa.Key points• First time critical review of the heterologous process for recombinant THCA/CBDA production and critical review of bottlenecks and limitations for a bioengineered technical process• Integrative approach of protein engineering, systems biotechnology, and biochemistry of yeast physiology and biosynthetic cannabinoid enzymes• Comparison of NphB and CsPT aromatic prenyltransferases as rate-limiting catalytic steps towards cannabinoids in yeast as platform organisms Graphical abstract.

Keywords: Bioengineering; Cannabidiol; Cannabinoids; Cannabis sativa; CsPT; Natural Product Biotechnology; NphB; Synthetic biology; Tetrahydrocannabinol.

Graphical abstract

Fig. 1

Tetrahydrocannabinolic acid (THCA) biosynthesic pathway in C. sativa L.. A total of six enzymes (AAE1, GPPS, OLS, OAC, CBGAS, THCAS) form THCA from isopentenyl diphosphate (IPP) and dimethylallylphosphate (DMAPP) synthesized in the MEP pathway, as well as hexanoic acid provided by the fatty acid biosynthesis

Fig. 2

Simulated intracellular concentrations of olivetolic acid pathway intermediates over a simulation time of 10 h. Hexanoic acid (HA, ), hexanoyl-CoA (HCoA, ), and olivetolic acid (OA, ) reach an intracellular non-toxic steady state

Fig. 3

Simulated intracellular concentrations of olivetolic acid (OA, ), geranyl pyrophosphate (GPP, ), and (CBGA, ) over a simulation time of 10 h. After several genetic optimizations and in contrast to the accumulation of GPP, OA reaches a stable equilibrium concentration. This implies that it is the limiting intermediate in the subsequent formation of CBGA

Fig. 4

Simulated intracellular concentrations of mevalonate pathway intermediates over a simulation time of 40 h. While farnesyl pyrophosphate (FPP, ) and geranyl pyrophosphate (GPP, ) accumulate, cannabigerolic acid (CBGA, ) reaches a steady state while being used as substrate itself. Neither isopentenyl pyrophosphate (IPP, ) nor dimethylallyl pyrophosphate (DMAPP, ) are detectable in significant amounts as they get converted into GPP/FPP efficiently

Fig. 5

Simulated intracellular concentrations of cannabinoid pathway intermediates and product over a simulation time of 40 h. From the precursors geranyl pyrophosphate (GPP, ) and olivetolic acid (OA, ), cannabigerolic acid (CBGA, ) is formed, which itself is converted to the product Δ⁹-tetrahydrocannabinolic acid (THCA, ). The remaining farnesyl pyrophosphate (FPP, ) concentration can be seen as potential to increase GPP production in future attempts

Fig. 6

Sensitivity analysis of the output THCA concentration. The bars represent the relative magnitude of THCA concentration increase when one intermediate is varied over time. Acetyl-CoA (ACCOA), cannabigerolic acid (CBGA), olivetolic acid (OA), geranyl pyrophosphate (GPP), isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, and mevalonate (MVA) are chosen as input intermediates to be varied in the analysis