Dual-Activity Polypeptides for Expanded Substrate Range in Biosynthesis
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Solution Overview
Problem
Biosynthetic thiolases have limitations such as inactivation by electrophilic substrates and inability to condense acetyl-CoA with substrates longer than 4 carbon atoms, restricting the diversity of chemicals that can be produced via 3-keto-acyl-CoA intermediates.
Innovation Solution
Development of polypeptides with dual CoA transferase and β-ketothiolase activities, classified under EC 2.8.3., which can catalyze CoA transfer and Claisen condensation to produce 3-keto-acyl-CoA esters from acetyl-CoA with alkanoic, alkenoic, hydroxy-, or halo-acids, enabling the production of longer chain 3-keto acids and derivatives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional biosynthetic thiolases are used, then C—C bond formation can occur, but the enzyme is inactivated by electrophilic substrates and cannot condense acetyl-CoA with substrates longer than 4 carbon atoms
Solution Approach 1:
The patent divides the functionality into separate enzymes: a thiolase for C—C bond formation and a CoA transferase for substrate activation. This segmentation allows each enzyme to be optimized for its specific function, avoiding the limitations of trying to make a single enzyme perform multiple functions with conflicting requirements
Solution Approach 2:
The patent introduces CoA transferase as an intermediary enzyme that activates substrates by transferring CoA before they enter the thiolase reaction. This intermediary step protects the thiolase from electrophilic substrate inactivation and enables the use of diverse substrates including those longer than 4 carbon atoms
2Productivity
If biosynthetic thiolases are used for C—C bond formation, then 3-keto-acyl-CoA intermediates can be produced, but the diversity of chemicals that can be produced is restricted
Solution Approach 1:
The patent creates a universal enzymatic system where the CoA transferase can process multiple types of substrates (alkanoic, alkenoic, hydroxy-, and halo-acids) and the thiolase can accept diverse CoA-activated substrates. This multi-functional system greatly expands the diversity of 3-keto-acyl-CoA intermediates and downstream chemicals that can be produced
3Adaptability or versatility
If separate enzymes are used for CoA transfer and Claisen condensation, then substrate range is expanded, but process complexity increases
Solution Approach 1:
The patent combines the CoA transferase and thiolase into a coupled enzymatic system where the products of one enzyme directly serve as substrates for the next. This merging of functions in sequence maintains substrate range expansion while managing complexity through efficient metabolic pathway integration
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 polypeptides overcome the limitations of traditional biosynthetic thiolases by enabling the production of a wider range of chemicals, including precursors for bulk chemicals, polyhydroxyalkanoate pathways, and clostridial fermentation products, by facilitating C—C bond formation and CoA transfer with various substrates.
Implementation Method 1
polypeptides with dual CoA transferase and β-ketothiolase activities, which can catalyze CoA transfer and Claisen condensation
Implementation Method 2
polypeptides with dual CoA transferase and β-ketothiolase activities, which can catalyze CoA transfer and Claisen condensation to produce 3-keto-acyl-CoA esters
Implementation Method 3
Biosynthetic thiolases catalyse carbon-carbon bond formation via a thioester-dependent Claisen-condensation reaction mechanism
Data Source
AI summary
This document describes polypeptides with dual CoA transferase and β-ketothiolase activities and variants thereof, use of such polypeptides in biosynthetic methods, and non-naturally occurring hosts comprising such polypeptides.