Variant Acyl-ACP Thioesterases for Fatty Acid Profile Control
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Solution Overview
Problem
Current methods for producing tailored oils with desired fatty acid profiles in oleaginous organisms are limited in their ability to precisely control acyl-ACP thioesterase substrate specificity, leading to suboptimal fatty acid profiles and reduced enzymatic activity.
Innovation Solution
Development of variant acyl-ACP thioesterases with specific N-terminal hydrophobic and specificity domains, combined with linker domains, to enhance enzymatic activity and alter fatty acid profiles by swapping and mutating domains, particularly incorporating amino acids like Asn91, Pro92, Val127, Leu133, and Ile163, to preferentially liberate C8, C10, C12, or C14 fatty acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If domain swapping and mutation methods are used to alter thioesterase substrate specificity, then fatty acid profile control is improved, but enzyme structural complexity increases
Solution Approach 1:
The thioesterase enzyme is divided into functional domains (N-terminal hydrophobic domain, specificity domain, C-terminal catalytic domain). By swapping only the N-terminal domains while retaining the catalytic domain, the invention achieves precise control over substrate specificity without redesigning the entire enzyme structure, thus improving fatty acid profile control while managing structural complexity.
Solution Approach 2:
The invention modifies only the local N-terminal region of the thioesterase enzyme that determines substrate specificity, rather than altering the entire enzyme. This localized modification approach allows precise control of fatty acid profiles while maintaining the core catalytic function and minimizing overall structural complexity.
2Reliability
If variant thioesterases with altered specificity domains are introduced, then enzymatic activity toward specific substrates is improved, but protein sequence complexity increases
Solution Approach 1:
The C-terminal catalytic domain is designed to be universal across different thioesterase variants, performing the core hydrolysis function. Only the N-terminal specificity domains are varied to target different substrates (C8:0, C10:0, C12:0, C14:0 acyl-ACPs). This modular universality improves enzymatic activity for specific substrates while reducing protein sequence complexity by reusing the same catalytic domain.
3Manufacturing precision
If precise control of acyl-ACP thioesterase substrate specificity is achieved through domain swapping, then fatty acid profile precision is improved, but method complexity increases
Solution Approach 1:
The method segments the enzyme modification process into domain selection, domain swapping, and expression steps. By treating the N-terminal domain as a separate interchangeable module, the method achieves precise fatty acid profile control through systematic domain exchange rather than complex whole-enzyme redesign, thereby managing method complexity.
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 variant acyl-ACP thioesterases demonstrate increased activity and altered specificity, enabling the production of novel or high-value commercial products with improved fatty acid profiles in oleaginous cells, such as microalgae, leading to enhanced lipid production and economic viability.
Implementation Method 1
the plant TEs have been shown to cluster into two families, FatAs, which show marked preference for 18:1-ACP with minor activity towards 18:0- and 16:0-ACPs; and FatBs, which hydrolyze primarily saturated acyl-ACPs
Data Source
AI summary
The present invention relates to variant thioesterases and their use in plants, e.g., to increase enzymatic activity and to promote increased production of mid-chain length fatty acids (e.g., 8 to 14 carbons) and at desired ratios. Further disclosed herein are methods of manufacturing renewable chemicals through the manufacture of novel triglyceride oils followed by chemical modification of the oils. Oils containing fatty acid chain lengths of C8, C10, C12 or C14 are also disclosed and are useful as feedstocks in the methods described herein.