Non-decarboxylative Thiolase Platform for Alpha-functionalized Synthesis
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Solution Overview
Problem
Current carbon-carbon bond forming reactions in natural biological systems, such as fatty acid and polyketide biosynthesis, are limited by the energy-intensive ATP-dependent 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, along with hydroxyacyl-CoA dehydrogenases, enoyl-CoA hydratases, and enoyl-CoA reductases, allowing for iterative chain elongation and termination to produce a wide range of alpha-functionalized products with varying functionalities and chain lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If decarboxylative Claisen condensation with malonyl thioesters is used, then carbon chain elongation can occur, 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 invention uses acetyl-CoA directly in thiolase-catalyzed condensation reactions, thereby 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 Claisen condensation (requiring malonyl-CoA) to non-decarboxylative thiolase-catalyzed condensation (using acetyl-CoA). This parameter change eliminates the need for ATP-dependent activation and enables direct use of acetyl-CoA as the extender unit
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 applies universality by designing a non-decarboxylative condensation platform that can accept diverse extender units with different functional groups (hydroxyl, amino, carboxyl, etc.) rather than being restricted to carboxylic groups. The thiolase enzyme system can process various acyl-CoA substrates, enabling synthesis of alpha-functionalized products with diverse functionalities
Solution Approach 2:
The patent changes the substrate acceptance parameters by using non-decarboxylative condensation chemistry that does not require carboxylic groups at the beta-site. This allows incorporation of extender units with different functional groups, thereby expanding product diversity while maintaining carbon chain elongation capability
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 approach enhances the energy efficiency and product diversity of carbon-chain elongation pathways, combining the versatility of biosynthetic pathways with the efficiency of fermentative processes, enabling the production of alpha-functionalized chemicals and fuels like carboxylic acids, alcohols, and hydrocarbons.
Implementation Method 1
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 beta-keto acyl-CoA
Implementation Method 2
subsequent beta-reduction reactions by hydroxyacyl-CoA dehydrogenases (HACDs), enoyl-CoA hydratases (ECHs) and enoyl-CoA reductases (ECRs) enable iteration
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.