Engineered Olivetolic Acid Cyclase Variants for Enhanced Cannabinoid Production
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Solution Overview
Problem
Current methods for producing cannabinoids, such as olivetolic acid, are limited by the efficiency and specificity of wild-type olivetolic acid cyclases, which restrict the production of desired compounds and their derivatives.
Innovation Solution
Engineered non-natural olivetolic acid cyclases with specific amino acid variations are developed to enhance catalytic activity, affinity, and stability, allowing for the production of hydroxylated and alkylated benzoic acid precursors that can be used to form various cannabinoids and their analogs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type olivetolic acid cyclase is used, then the enzyme maintains natural substrate specificity, but the catalytic activity and production rate of desired compounds are limited
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues in the OAC enzyme sequence to alter enzyme properties. Specific mutations at positions such as 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 change the enzyme's catalytic properties, substrate binding affinity, and thermal stability, thereby improving productivity while maintaining controlled specificity
Solution Approach 2:
The patent applies local quality by making specific localized changes to the enzyme structure through point mutations at defined amino acid positions. Each mutation affects a specific local region of the enzyme that interacts with substrates or cofactors, allowing optimization of catalytic activity and stability without completely redesigning the entire enzyme structure
2Productivity
If wild-type olivetolic acid cyclase is used, then the enzyme structure remains simple and well-characterized, but the catalytic efficiency and affinity for substrates are insufficient
Solution Approach 1:
The patent systematically changes enzyme parameters through amino acid substitutions, deletions, and insertions to optimize catalytic efficiency. The modifications are applied to specific positions in the enzyme sequence to enhance substrate binding affinity, catalytic rate, and overall efficiency while tracking the impact on enzyme complexity
3Productivity
If wild-type olivetolic acid cyclase is used, then the enzyme expression is straightforward, but the production yield and activity level are limited
Solution Approach 1:
The patent modifies enzyme parameters including expression levels, protein stability, and folding efficiency through amino acid mutations. These changes improve production yield by enhancing enzyme stability during expression and purification while maintaining reasonable manufacturing complexity
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 engineered cyclases increase the rate and specificity of olivetolic acid production, enabling the formation of diverse cannabinoid compounds with improved activity and expression, overcoming limitations of wild-type enzymes.
Implementation Method 1
OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to DABB-type polyketide cyclase enzymes from Streptomyces and to stress-responsive proteins in plants
Data Source
AI summary
Described herein are non-natural olivetolic acid cyclases (OAC) variants capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate at a greater rate than a wild type or control OAC. The non-natural OAC (and OLS) can be expressed in an engineered cell having a pathway to form cannabinoids, which include CBGA, its analogs and derivatives. CBGA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.