Recombinant BBE Polypeptides for Soluble Cannabinoid Biosynthesis
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Solution Overview
Problem
Current methods for biosynthesizing cannabinoids rely on enzymes from Cannabis sativa, which cannot be expressed in a soluble form in E. coli systems, limiting their use in in vitro cannabinoid biosynthesis.
Innovation Solution
Development of recombinant polypeptides with berberine bridge enzyme (BBE) activity derived from bacterial host organisms, such as Phytohabitans suffuscus and Streptomyces species, which can catalyze the oxidative cyclization of prenylated compounds like CBGA to produce cannabinoids like CBCA, THCA, and CBDA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plant-derived cannabinoid synthases (THCAS, CBDAS, CBCAS) from Cannabis sativa are used for oxidative cyclization of CBGA, then the desired cannabinoid products (THCA, CBDA, CBCA) can be produced, but these enzymes cannot be expressed in soluble form in E. coli systems, limiting their use in in vitro biosynthesis
Solution Approach 1:
The patent introduces bacterial BBE homologs (Clz9 from Streptomyces sp. CNH287, SfBBE from Phytohabitans suffuscus, and other Streptomyces species) as intermediary enzymes that can perform the same oxidative cyclization function as plant synthases but are compatible with E. coli expression systems. These bacterial enzymes serve as mediators between the desired biochemical transformation and the practical requirements of recombinant protein production in E. coli, achieving both functional equivalence and expression solubility
Solution Approach 2:
The patent changes the source organism parameter from plant (Cannabis sativa) to bacterial (Streptomyces sp., Phytohabitans suffuscus) to obtain enzymes with different biochemical properties that are soluble in E. coli. This parameter change in enzyme source allows the same catalytic function to be achieved while resolving the solubility and expression issues associated with plant-derived synthases
2Reliability
If bacterial BBE homologs (e.g., Clz9 from Streptomyces sp. CNH287) are used for oxidative cyclization, then soluble expression in E. coli and increased CBCA selectivity are achieved, but the enzyme sequence identity to plant synthases is very low
Solution Approach 1:
Instead of trying to modify plant synthases to achieve solubility (the conventional approach), the patent inverts the strategy by selecting bacterial enzymes that naturally possess both the required catalytic activity and solubility properties. This inversion of the selection criterion - from starting with plant enzymes and attempting modification to starting with bacterial enzymes and selecting for desired properties - enables discovery of enzymes with low sequence identity but high functional performance and CBCA selectivity
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 recombinant polypeptides demonstrate increased selectivity and activity in converting CBGA to CBCA, overcoming the limitations of plant-derived enzymes and enabling efficient cannabinoid biosynthesis in cell-free systems.
Implementation Method 1
the recombinant polypeptides with BBE activity of the present disclosure can be substituted for the various C. sativa synthase and carry out this final step of oxidative cyclization in such cell-free biosynthetic systems
Data Source
AI summary
The application relates to recombinant polypeptides having berberine bridge enzyme (BBE) activity that are derived from microorganisms, such as Phytohabitans sufuscus, Streptomyces sp. AJS327, Streptomyces varsoviensis, Actinomadura pelletieri, Streptomyces flaveolus, Streptomyces sp. Ru71. or Streptomyces sp. CNH287, and the use of these recombinant polypeptides in compositions and methods for the oxidative cyclization of prenylated compounds, such as CBGA, in carrying out the biosynthesis, including cell-free biosynthesis, of cyclized cannabinoid compounds, such as CBCA, THCA, and CBDA.