Cannabinoid fermentation is a new, challenging, biosynthetic method developed as alternative to cannabis-derived extractions. This technique is not dependent on cultivating the plant or influenced by secondary metabolite variations during the plant life-cycle due to environmental stresses such as pests, weather, and so on. [1]
Metabolic engineering-derived cannabinoid production permits the decrease of economic costs, in addition to reduced use of pesticides, water, land, and solvents employed during the isolation and purification processes. Through the action of recombinant bacteria and yeasts, it is possible to reproduce the plant metabolic pathways responsible for cannabinoid biosynthesis.
The heterologous production of cannabinoids in host microorganisms is linked to their genetic tools capacity and the choice of a suitable substrate for the growth and proliferation of the chosen cell-factory. Escherichia coli, Saccharomyces cerevisiae, Komagataella phaffii (Pichia pastoris), and Kluyveromyces marxianus are among the most studied microorganisms used in the production of cannabinoids such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD), ∆9-tetrahydrocannabivarin (∆9-THCV), etc. While in the agricultural-based method, the amount and variety of compounds produced is restricted by the plant genetics and selective breeding, microorganism-derived cannabinoid formation can be guided depending on the tailored pathways and the introduced genes and enzymes.
The recent work of Luo et al. [2] can be taken as example to understand the ratio of heterologous cannabinoid biosynthesis. The process starts by growing S. cervisiae yeast in a simple sugar, galactose substrate. Because cannabinoid biosynthesis in plants starts with metabolic pathways to produce geranyl pyrophosphate (GPP) and olivetolic acid (OA) [1], the native S. cervisiae mevalonate pathway was engineered to improve GPP production. The synthesis of OA was increased by introducing cannabis genes encoding OA and by feeding the yeast with hexanoic acid which can be converted to hexanoyl-CoA by an endogenous acyl activating enzyme (AAE). [2]
Once reasonable amounts of cannabinoid precursors GPP and OA were reached, the cannabis-derived enzyme geranylpyrophosphate:olivetolate geranyltransferase (GOT) was introduced to synthesize cannabigerolic acid (CBGA), the precursor of all cannabinoids. Through the introduction of enzymes such as tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase, and cannabichromenic acid (CBCA) synthase, it was possible to obtain the corresponding acidic cannabinoids, promoting the cyclization of the monoterpene moiety in CBGA. [1] By replacing hexanoic acid with butanoic acid, it is also possible to obtain the biosynthetic pool to produce propyl cannabinoids such as tetrahydrocannabivarinic acid (THCVA) and cannabidivarinic acid (CBDVA), meaning that by changing the feeding substrate, it is possible to alter the cannabinoid moiety responsible for different receptor binding affinity and potency. [2]
This innovative method for cannabinoid biosynthesis can overcome the issues linked to cannabis production in those places where it is still illegal and it can enhance the production of minor canabinoids that are difficult to recover through extraction. Nevertheless, the variety of heterologous-produced cannabinoids is limited to the biosynthetic pool available during the fermentation process and through this method, it is not possible to obtain a full spectrum extract including other compounds such as terpenes and flavonoids that contribute to the therapeutic effect of cannabis.
References:
[1] Favero GR, de Melo Pereira GV, de Carvalho JC, de Carvalho Neto DP, Soccol CR. Converting sugars into cannabinoids—The state-of-the-art of heterologous production in microorganisms. Fermentation. 2022; 8(2):84. [journal impact factor = 5.123; times cited = 2][2] Luo X, Reiter MA, d’Espaux L, et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast [published correction appears in Nature. 2020 Apr;580(7802):E2]. Nature. 2019;567(7746):123-126. [journal impact factor = 69.504; times cited = 372]
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