The Biochemistry of Phytocannabinoids and Metabolic Engineering of Their Production in Heterologous Systems

Abstract

The medicinal properties of cannabis and the its legal status in several countries and jurisdictions has spurred the massive growth of the cannabis economy around the globe. The value of cannabis stems from its euphoric activity offered by the unique phytocannabinoid tetrahydrocannabinol (THC). However, this is rapidly expanding beyond THC owing to other non-psychoactive phytocannabinoids with new bioactivities that will contribute to their development into clinically useful drugs. The discovery of the biosynthesis of major phytocannabinoids has allowed the exploration of their heterologous production by synthetic biology, which may lead to the industrial production of rare phytocannabinoids or novel synthetic cannabinoid pharmaceuticals that are not easily offered by cannabis plants. This review summarizes the biosynthesis of major phytocannabinoids in detail, the most recent development of their metabolic engineering in various systems, and the engineering approaches and strategies used to increase the yield.

Keywords: FAD-dependent monooxygenase; aromatic prenyltransferase; cannabinoid biosynthesis; metabolic engineering; polyketide synthase; synthetic biology; yeast fermentation.

Figure 1

Representative structures for endocannabinoids, phytocannabinoids, intermediates and numbering system.

Figure 2

The native and engineered biosynthetic pathway for phytocannabionids in C. sativa. Enzymes in italics are mutant enzymes. The abbreviations are listed as follows. The cannabinoid pathway: TKS, tetraketide synthase; OAC, olivetolic acid synthase; PKS, polyketide synthase; NphB, naphterpin biosynthetic cluster gene B; PT, prenyltransferase; THCAS, tetrahydrocannabinolic acid synthase; CBDAS, cannabidiolic acid synthase; CBCAS, cannabichromenic acid synthase. MVA pathway: mvaE, acetoacetyl-CoA thiolase/HMG-CoA reductase; mvaS, HMG-CoA synthase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, diphosphomevalonate decarboxylase; IDI1 isopentenyl diphosphate:dimethylallyl diphosphate isomerase; ERG20, farnesyl pyrophosphate synthetase. MEP pathway: DXS, deoxyxylulose 5-phosphate synthase; DXR, deoxyxylulose 5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthase; IspE, CDP-ME kinase; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; IspH, HMB-PP reductase. Fatty acid biosynthesis: BktB, β-keto thiolase; ACC, acetyl CoA carboxylase; MCT1: malonyl CoA-acyl carrier protein transacylase; HBD/PaaH1, β-hydroxybutyryl-CoA de-hydrogenase; CRT: 3-hydroxybutyryl-CoA dehydratase; Ter, trans-enoyl-CoA reductase; FAA2, Long-chain-fatty-acid--CoA ligase 2; KmTES1, Kluyveromyces marxianus acyl-CoA thioesterase; AAE: acyl activating enzyme. Species abbreviations: Cs, Cannabis sativa; St, Streptomyces strain CL109; Di, Dictyostelium discoideum; Ef, Enterococcus faecalis; Re, Ralstonia eutropha; Ca, Clostridium acetobutylicum; Td, Treponema denticola; Sc, Saccharomyces cerevisiae; Km, Kluyveromyces marxianus.