Chimeric DGAT1 Proteins for Enhanced TAG Production
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Solution Overview
Problem
Current methods for increasing triacylglyceride (TAG) production in plants face challenges due to the enzyme diacylglycerol-O-acyltransferase (DGAT) having the lowest specific activity in the Kennedy pathway, making it a bottleneck, and previous attempts to improve DGAT1 through biotechnological means have shown limited success.
Innovation Solution
The development of chimeric DGAT1 proteins with enhanced activity, stability, and altered oligomerization properties by combining N-terminal and C-terminal portions of different DGAT1 proteins, specifically designing the junction between these portions to be upstream of the first transmembrane domain, resulting in increased TAG production and altered lipid profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DGAT1 enzyme is used for TAG synthesis, then TAG production occurs, but the specific activity is low making it a bottleneck in the pathway
Solution Approach 1:
The patent combines N-terminal and C-terminal portions of different DGAT1 proteins from plant species to create chimeric DGAT1 proteins. This merging of functional domains from different sources aims to achieve synergistic effects that overcome the low specific activity limitation of individual DGAT1 enzymes while maintaining their TAG synthesis function.
Solution Approach 2:
The patent creates variant chimeric DGAT1 proteins with different amino acid sequences by modifying the combination of N-terminal and C-terminal portions. These parameter changes in the enzyme structure are designed to improve catalytic efficiency and specific activity, thereby resolving the bottleneck in TAG production rate.
2Reliability
If chimeric DGAT1 proteins are designed by combining N-terminal and C-terminal portions, then enzyme activity and stability are enhanced, but the protein structure complexity increases
Solution Approach 1:
The patent segments the DGAT1 protein into distinct N-terminal and C-terminal functional domains that can be independently selected and recombined. This segmentation allows for systematic creation of chimeric proteins with controlled complexity, where each domain contributes specific functional properties to enhance overall enzyme stability and activity.
3Productivity
If junction between N-terminal and C-terminal portions is placed upstream of the first transmembrane domain, then TAG production increases, but the risk of disrupting transmembrane function increases
Solution Approach 1:
The patent positions the junction between N-terminal and C-terminal portions upstream of the first transmembrane domain, using this specific location as an intermediary zone that allows recombination without disrupting the critical transmembrane anchoring function. This strategic positioning enables enhanced TAG production while preserving the essential membrane association required for enzyme function.
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 chimeric DGAT1 proteins demonstrate increased activity and stability, leading to enhanced TAG production, with some variants showing up to 150% more TAG production compared to control cells, and altered lipid profiles, such as altered proportions of 16:0, 18:0, and 18:1 fatty acids in triacylglycerols.
Implementation Method 1
DGAT is termed as the committed triacylglycerol-forming enzyme. DAG is located at the branch point between TAG and membrane phospholipid biosyntheses, DGAT potentially plays a decisive role in regulating the formation of TAG in the glycerolipid synthesis pathway
Data Source
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AI summary
The invention provides chimeric DGAT1 proteins comprising: a) at their N-terminal ends, an N- terminal portion of a first DGAT1 protein, and b) at their C-terminal ends, a C-terminal portion of a second DGAT1 protein. The chimeric DGAT proteins show enhanced activity relative to at least one of the first DGAT1 protein and the second DGAT1 protein. The chimeric DGAT proteins of the invention can be expressed in cells to increase cellular lipid accumulation and/or modify the cellular lipid profile. The invention also provides polynucleotides encoding the chimeric DGAT1 proteins, cells and compositions comprising the polynucleotides or chimeric DGAT1 proteins, and methods using the chimeric DGAT1 proteins to produce oil.