DGAT1 N-Terminal Mutations for Stable Lipid Production
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Solution Overview
Problem
Current methods for increasing triacylglycerol (TAG) production in plants and microorganisms face limitations, particularly due to the enzyme diacylglycerol-O-acyltransferase (DGAT) being a bottleneck with low specific activity and challenges in preventing TAG catabolism, especially in non-oleaginous tissues and across various developmental stages.
Innovation Solution
Modification of the N-terminal region of DGAT1 proteins upstream of the acyl-CoA binding site to enhance activity, stability, and substrate specificity, including truncations and additions such as a flexible peptide linker, to increase cellular lipid accumulation without reducing protein accumulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DGAT enzyme activity is increased to boost triacylglycerol production, then lipid accumulation is improved, but protein accumulation decreases due to reduced stability
Solution Approach 1:
The patent modifies the N-terminal region of DGAT1 proteins by changing amino acid sequences (parameter changes) to create variants with both increased enzymatic activity and improved stability. Specific mutations in the N-terminal region alter protein folding and interaction properties, allowing the enzyme to maintain high activity while accumulating to higher levels in cells.
Solution Approach 2:
The invention focuses modifications on the N-terminal region of the DGAT1 protein, which is a specific local area, to improve overall protein properties. By targeting the N-terminal region for specific amino acid changes, the patent achieves localized optimization that enhances both activity and stability without affecting the entire protein structure.
2Productivity
If DGAT1 protein levels are increased to enhance lipid production, then triacylglycerol accumulation is improved, but enzyme activity decreases due to reduced specific activity
Solution Approach 1:
The patent employs parameter changes by modifying the N-terminal amino acid sequence of DGAT1 to create enzymes with higher specific activity. These mutations optimize the enzyme's catalytic efficiency, allowing fewer protein molecules to achieve the same lipid production level, thereby maintaining high enzyme activity even at moderate protein levels.
Solution Approach 2:
The invention applies local quality by concentrating functional improvements in the N-terminal region of the protein. This localized modification optimizes the enzyme's active site accessibility and substrate binding, enhancing specific activity without requiring widespread changes throughout the protein structure.
3Productivity
If conventional DGAT1 is used to increase oil content, then lipid production is improved, but protein accumulation is reduced in non-oleaginous tissues
Solution Approach 1:
The patent uses parameter changes by creating N-terminal mutants of DGAT1 that have altered protein stability and accumulation properties. These mutations prevent rapid degradation of the enzyme in non-oleaginous tissues, allowing the protein to accumulate to higher levels and maintain activity across different tissue types and developmental stages.
Solution Approach 2:
The invention achieves universality by creating a DGAT1 variant that functions effectively across multiple tissue types and developmental stages. The N-terminal modifications enable the enzyme to be stable and active not only in oleaginous tissues but also in non-oleaginous tissues, providing broad applicability for increasing lipid production throughout the plant.
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 modified DGAT1 proteins demonstrate increased activity and stability, leading to enhanced lipid production in cells and plants, with altered lipid profiles and increased oil content, overcoming the limitations of previous approaches.
Implementation Method 1
The PA is then dephosphorylated by the enzyme phosphatidic acid phosphatase (PAP; EC3.1.3.4) to release the immediate precursor for TAG, the sn-1,2-diacylglycerol (DAG). Finally, DAG is acylated in the sn-3 position by the enzyme diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) to form TAG.
Data Source
AI summary
The invention provides modified DGAT1 proteins that are modified in the N-terminal region upstream of the acyl-Co A binding site. The modified DGAT proteins show enhanced activity, without reduced protein accumulation when expressed in cells. The modified DGAT1 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 modified DGAT1 proteins, cells and compositions comprising the polynucleotides or modified DGAT proteins, and methods using the modified DGAT1 proteins to produce oil.