Cannabinoid Synthase Variants Without Disulfide Bonds for Microbial Production
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Solution Overview
Problem
Existing cannabinoid synthases in microbial organisms like Escherichia coli lack efficient mechanisms for forming disulfide bonds, limiting the production of cannabinoids such as THCA, CBDA, and CBCA, which are valuable for their therapeutic properties.
Innovation Solution
Development of non-natural cannabinoid synthases with specific amino acid variations, specifically in THCAS, CBDAS, and CBCAS, that lack a disulfide bond between alpha helices αA and αC, enabling these enzymes to catalyze the oxidative cyclization of CBGA into THCA, CBDA, and CBCA, respectively, in microbial hosts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild type cannabinoid synthases are used in microbial hosts, then the enzymes can catalyze oxidative cyclization of CBGA, but the production is limited due to inability to form disulfide bonds in microbial cytoplasm
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of cannabinoid synthases to eliminate cysteine residues that form disulfide bonds (e.g., C37A/C99A mutations in THCAS). This structural parameter change allows the enzyme to function in microbial cytoplasm where disulfide bond formation is limited, thereby improving productivity in microbial hosts without sacrificing catalytic activity.
Solution Approach 2:
The patent creates modified copies of wild type cannabinoid synthases with specific amino acid substitutions. These copied enzymes retain the catalytic function of the original enzymes but have altered structural features (removed disulfide bonds) that enable them to function properly in microbial hosts, thus resolving the contradiction between maintaining enzyme functionality and achieving high productivity.
2Stability of the object's composition
If disulfide bonds are formed in wild type synthases, then structural stability is maintained, but the enzymes cannot function efficiently in microbial organisms lacking disulfide bond formation mechanisms
Solution Approach 1:
The patent changes the structural parameters of the enzyme by removing disulfide bonds through amino acid substitution (e.g., replacing cysteine with alanine at positions 37 and 99). This parameter change allows the enzyme to maintain stability through alternative structural mechanisms while enabling function in microbial hosts, thereby resolving the contradiction between structural stability and productivity in microbial systems.
Solution Approach 2:
The patent introduces an intermediary solution by creating a modified enzyme variant that serves as a bridge between the structural requirements of the wild type enzyme and the functional requirements of microbial hosts. The modified synthase acts as an intermediary structure that achieves both stability and functionality in the microbial environment.
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
These modified synthases enhance the production of cannabinoids like THCA, CBDA, and CBCA in microbial hosts, overcoming the limitations of natural synthases and facilitating the production of therapeutic compounds.
Implementation Method 1
wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid
Data Source
AI summary
The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase Δ9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (αA, αB and αC) where a disulfide bond is not formed between alpha helix αA and alpha helix αC, wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid. The invention further relates to a non-natural Δ9-tetrahydrocannabinolic acid synthase (THCAS), a non-natural cannabidiolic acid synthase (CBDAS), and a non-natural cannabichromenic acid synthase (CBCAS) comprising at least one amino acid variation as compared to a wild type THCAS, CBDAS, or CBCAS, respectively, comprising three alpha helices (αA, αB and αC) and wherein a disulfide bond is not formed between alpha helix αA and alpha helix αC. The invention also relates to a nucleic acid, expression construct, and engineered cell for making the non-natural THCAS, CBDAS, and/or CBCAS. Also provided are compositions comprising the non-natural THCAS, CBDAS, and/or CBCAS; isolated non-natural THCAS, CBDAS, and/or CBCAS enzymes; methods of making the isolated enzymes; cell extracts comprising cannabinoids; and methods of making cannabinoids.