Yeast Phytocannabinoid Production via Enzyme Substitution
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Solution Overview
Problem
The production of phytocannabinoids in Cannabis sativa is labor and energy intensive, and the cultivation of the plant is costly and heavily regulated, while chemical synthesis of phytocannabinoid analogues is also labor-intensive, necessitating a more efficient and scalable method for their production.
Innovation Solution
Transgenic Saccharomyces cerevisiae yeast cells are transformed with genes coding for specific enzymes such as NphB prenyltransferase and polyketide synthases like DiPKS, enabling the synthesis of phytocannabinoids and analogues like cannabigerolic acid and cannabigerol without the need for hexanoic acid, which is toxic to yeast, and allowing for increased production of malonyl-CoA and geranyl pyrophosphate precursors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If Cannabis sativa is cultivated for phytocannabinoid production, then phytocannabinoids can be obtained, but the process becomes labor and energy intensive with high costs and heavy regulation
Solution Approach 1:
The patent uses yeast (Saccharomyces cerevisiae) as an intermediary organism to produce phytocannabinoids through heterologous expression of plant enzymes. The yeast serves as a mediator that converts plant-derived precursors (olivetolic acid and GPP) into phytocannabinoids, eliminating the need for direct Cannabis cultivation while maintaining production capability
Solution Approach 2:
The patent copies the phytocannabinoid biosynthetic pathway from Cannabis sativa into yeast by introducing heterologous genes encoding plant enzymes (OAS, OAC, and prenyltransferase). This creates a synthetic biological system that replicates the natural plant pathway in a more controllable and scalable host organism
2Quantity of substance
If chemical synthesis is used to produce phytocannabinoid analogues, then production can be achieved, but the process becomes labor intensive and costly
Solution Approach 1:
The patent replaces chemical synthesis mechanisms with biological catalysis. Instead of using chemical reagents and multi-step synthetic procedures, the system employs enzymatic catalysis by heterologously expressed plant enzymes in yeast, which naturally perform the same transformations under mild physiological conditions
Solution Approach 2:
The yeast system performs self-service by autonomously carrying out the biosynthetic transformations. The introduced enzymatic pathway enables the yeast to automatically convert available precursors into phytocannabinoid analogues through its metabolic machinery, eliminating the need for manual chemical synthesis operations
3Quantity of substance
If C. sativa prenymltransferase is expressed in S. cerevisiae, then CBGa synthesis can occur, but the membrane-bound enzyme complicates expression and reduces production levels
Solution Approach 1:
The patent extracts the prenymltransferase catalytic function from its native membrane-bound context in Cannabis and implements it as a cytosolic enzyme in yeast. By using a bacterial prenymltransferase (NphB from Streptomyces coelicolor) that naturally functions in the cytosol, the system eliminates the complexity of membrane anchoring while preserving the essential catalytic activity for CBGa production
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enhances the yield and efficiency of phytocannabinoid production in yeast, reducing the need for costly plant cultivation and chemical synthesis, while avoiding the toxicity of hexanoic acid, thereby providing a scalable and cost-effective route to these pharmacologically active molecules.
Implementation Method 1
AltPT catalyzes synthesis of cannabigerolic acid (CBGa) from olivetolic acid and geranyl pyrophosphate (GPP)
Implementation Method 2
AltPT also catalyzes synthesis of cannabigerol (CBG) from olivetol and GPP
Implementation Method 3
a gene coding for the polyketide synthase CDS that catalyzes the synthesis of olivetolic acid from malonyl-CoA and hexanoyl-CoA
Data Source
AI summary
A method and cell line for producing phytocannabinoids and phytocannabinoid analogues in yeast. The method applies, and the cell line includes, a yeast cell transformed with a polyketide synthase CDS and a cytosolic prenyltransferase CDS. The polyketide synthase enzyme catalyzes synthesis of olivetol or methyl-olivetol, and may include Cannabis sativa olivetolic acid synthase or Dictyostelium discoideum polyketide synthase (“DiPKS”). The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol. The prenyltransferase enzyme catalyzes synthesis of cannabigerol or a cannabigerol analogue, and may include an αββα cytosolic prenyltransferase enzyme from Streptomyces sp CL190. The yeast cell may be modified to mitigate depletion of geranyl pyrophosphate for increasing available geranyl pyrophosphate for prenylation.