Engineered Prenyltransferases for CBGA Synthesis
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Solution Overview
Problem
Current methods for mass-producing specific phytocannabinoids like THCA, CBDA, and CBCA from cannabigerolic acid (CBGA) are limited by low biosynthesis rates and high byproduct formation, which hampers their therapeutic and recreational applications.
Innovation Solution
Development of novel CBGA and cannabigerovarinic acid synthases, along with recombinant membrane-bound prenyltransferases and soluble aromatic prenyltransferases, that enhance the synthesis of CBGA and CBGVA by optimizing prenyltransferase activity, selectivity, and intracellular availability of precursors, and using fusion proteins with polyketide cyclase and geranyl diphosphate synthase activities to increase titer and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native prenymltransferase is used for CBGA synthesis, then the biosynthesis pathway is naturally maintained, but the production rate is low and byproduct formation is high
Solution Approach 1:
The patent modifies the prenymltransferase enzyme through amino acid substitutions (e.g., F66L, Y139F, F154L mutations) to change its catalytic parameters, specifically improving substrate selectivity for GPP over FPP and enhancing conversion efficiency of OA to CBGA, thereby simultaneously increasing production rate and purity
Solution Approach 2:
The patent creates recombinant copies of prenymltransferase with optimized sequences based on natural variants (MPT1, MPT4, MPT21, MPT26, MPT31) and expresses them in heterologous host cells to achieve high-level CBGA production with improved selectivity, effectively copying and optimizing the natural enzyme function
2Quantity of substance
If aromatic acid importers are overexpressed to increase intracellular OA availability, then CBGA production potential increases, but byproduct formation from FPP competition increases
Solution Approach 1:
The patent introduces modified prenymltransferase as an intermediary enzyme that specifically channels the increased OA flux toward CBGA production by exhibiting high selectivity for GPP over FPP as prenyl donors, thereby mediating the conversion of OA to CBGA while minimizing competing reactions that produce byproducts
Solution Approach 2:
The patent changes the substrate specificity parameters of prenymltransferase through directed evolution and rational design, creating variants with enhanced preference for GPP over FPP, which allows high OA availability to be converted efficiently to CBGA without significant byproduct formation from FPP competition
3Productivity
If fusion proteins are used to improve enzyme stability and activity, then CBGA synthesis efficiency increases, but protein complexity increases
Solution Approach 1:
The patent merges multiple functional domains into fusion proteins, including prenymltransferase with signal peptides for membrane targeting, affinity tags for purification, and complementary enzyme activities, to create multifunctional constructs that improve stability, solubility, and catalytic efficiency while enabling simplified purification and localization
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
The approach significantly increases the production of CBGA and CBGVA while reducing byproduct formation, thereby enhancing the yield and purity of these cannabinoids, addressing the limitations of existing biosynthesis methods.
Implementation Method 1
novel CBGA and cannabigerovarinic acid (CBGVA) synthases and methods for improvement of their overall activities for the synthesis of CBGA and CBGVA from their respective precursors, olivetolic acid (OA) or divarinic acid (DVA) and geranyl pyrophosphate (GPP)
Implementation Method 2
by providing a fusion protein which contains a polyketide cyclase (PKC) in addition to a prenymltransferase and a GPP synthase, the flux of OA/DVA made from hexanoic or butyric acid is increased
Implementation Method 3
Fusions of CBGA synthases with other enzymes such as GPP synthases and their mutants
Data Source
AI summary
Disclosed herein are novel CBGA and CBGVA synthases and methods for improvement of their overall activities for the synthesis of CBGA and CBGVA from their respective precursors, olivetolic acid (OA) or divarinic acid (DVA) and GPP. Also disclosed are fusion proteins to enhance synthesis of CBGA and CBGVA. The methods described herein also increase the titer and the purity of CBGA and CBGVA made by a cell by 1) decreasing the formation of byproducts FCBGA and FCBGVA that are synthesized from the respective prenylation of OA and DVA with FPP, and/or 2) increasing the intracellular availability of OA and DVA.