Iterative Alpha-Functionalized Product Synthesis Platform
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Solution Overview
Problem
Natural carbon-carbon bond forming reactions in biological systems, such as fatty acid and polyketide biosynthesis, are limited by the energy-intensive activation of malonyl-CoA and the restricted range of extender units due to decarboxylative Claisen condensation mechanisms, which restricts the diversity of products that can be generated.
Innovation Solution
A CoA-dependent carbon elongation platform using de novo thiolase-catalyzed non-decarboxylative Claisen condensation that accepts functionalized primers and extender units, including alpha-functionalized acetyl-CoA, enabling iterative chain elongation with thiolases, dehydrogenases, dehydratases, and reductases to produce a wide range of alpha-functionalized products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If decarboxylative Claisen condensation with malonyl thioesters is used, then carbon chain elongation can proceed, but energy efficiency deteriorates due to ATP-dependent activation of acetyl-CoA to malonyl-CoA
Solution Approach 1:
The patent inverts the conventional decarboxylative Claisen condensation approach by using non-decarboxylative condensation with acetyl-CoA as the extender unit. Instead of activating acetyl-CoA to malonyl-CoA (which consumes ATP), the system directly uses acetyl-CoA in thiolase-catalyzed condensation reactions, eliminating the energy-intensive activation step while maintaining carbon chain elongation capability
Solution Approach 2:
The patent changes the chemical parameters of the condensation reaction by switching from decarboxylative to non-decarboxylative mechanism. This parameter change allows the use of acetyl-CoA instead of malonyl-CoA as the extender unit, fundamentally altering the energy requirements and enabling more energy-efficient carbon chain elongation
2Productivity
If decarboxylative Claisen condensation is used, then carbon chain elongation occurs, but product diversity deteriorates due to restriction of extender units to those with carboxylic groups at the beta-site
Solution Approach 1:
The patent makes the thiolase enzyme universal by enabling it to accept a wide variety of extender units beyond just malonyl-CoA derivatives. The non-decarboxylative mechanism allows acetyl-CoA and other acyl-CoA derivatives to serve as extender units, making the carbon chain elongation system multi-functional and capable of producing diverse alpha-functionalized products
Solution Approach 2:
By inverting from decarboxylative to non-decarboxylative condensation, the patent removes the structural restriction that required carboxylic groups at the beta-site of extender units. This inversion expands the range of acceptable extender units to include various alpha-functionalized acyl-CoA derivatives, thereby increasing product diversity
3Use of energy by moving object
If reverse beta-oxidation with thiolases is used, then energy efficiency improves by using acetyl-CoA directly, but product functionality deteriorates because only acetyl-CoA can serve as extender unit
Solution Approach 1:
The patent extends the universality of thiolase enzymes to accept multiple types of acyl-CoA derivatives as extender units, not just acetyl-CoA. This multi-functionality allows the system to maintain energy efficiency while producing alpha-functionalized products with diverse functionalities including carboxylic acids, alcohols, hydrocarbons, and amines
Solution Approach 2:
The patent changes the substrate specificity parameters of the thiolase enzyme system to accommodate various alpha-functionalized acyl-CoA derivatives. By modifying the enzyme's acceptance criteria for extender units, the system maintains the energy-efficient non-decarboxylative mechanism while expanding product functionality
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 platform achieves high product diversity while maintaining energy efficiency, combining the benefits of biosynthetic pathways with fermentative processes, allowing for the production of alpha-functionalized carboxylic acids, alcohols, hydrocarbons, and amines with varying functionalities.
Implementation Method 1
thiolases catalyze the non-decarboxylative Claisen condensation in which acetyl-CoA, instead of malonyl thioesters, serves as the extender unit
Implementation Method 2
subsequent beta-reduction reactions by hydroxyacyl-CoA dehydrogenases (HACDs)
Implementation Method 3
enoyl-CoA hydratases (ECHs)
Implementation Method 4
enoyl-CoA reductases (ECRs)
Data Source
AI summary
The use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing an iterative carbon chain elongation pathway that uses functionalized extender units. The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized β-keto acyl-CoA. Dehydrogenase converts alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratase converts alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned alpha-functionalized extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different β-reduction degree.