AED3-1 Protease Inhibition for Higher Plant Protein Yield
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Solution Overview
Problem
The widespread use of plant-based pharming technology for producing heterologous proteins is limited by the plant's natural defense responses, including transcriptional activation of defense genes, increased H2O2 production, and unintended proteolysis, which reduce biological output.
Innovation Solution
Inhibiting aspartic protease AED3-1 in plants by introducing nucleic acid molecules encoding inhibitors, such as shRNA, or through genomic modifications to reduce or eliminate AED3-1 function, combined with introducing nucleic acid molecules encoding heterologous proteins operably linked to promoters, enhances protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plant-based pharming technology is used to produce heterologous proteins, then cost-effectiveness and manufacturing scalability are improved, but the plant's natural defense responses cause unintended proteolysis that reduces protein yield
Solution Approach 1:
The patent extracts and targets the specific harmful function of AED3-1 protease while preserving the plant's natural defense mechanisms. By selectively inhibiting only this particular protease enzyme through RNAi or chemical inhibitors, the patent removes the harmful proteolytic activity without eliminating the plant's overall immune response capability, thus resolving the contradiction between maintaining plant defenses and preventing protein degradation.
Solution Approach 2:
The patent changes the functional state of the AED3-1 protease from active to inhibited through multiple approaches including RNA interference, chemical inhibitors, and genomic modifications. This parameter change in enzyme activity allows the plant to maintain its defensive functions while preventing the harmful proteolysis of heterologous proteins, thereby improving protein yield without compromising plant security.
2Reliability
If the plant's natural defense responses are activated to combat pathogenic invasion, then plant security is improved, but transcriptional activation of defense genes and H2O2 production reduce biological output
Solution Approach 1:
The patent segments the plant's defense response system by identifying and targeting specific components (AED3-1 protease) that cause harmful effects, while leaving other defense mechanisms intact. This segmentation allows the plant to maintain overall security through transcriptional activation of defense genes and H2O2 production, while specifically neutralizing the proteolytic threat to heterologous proteins.
Solution Approach 2:
The patent converts the harmful proteolytic activity of AED3-1 into a beneficial control mechanism by using RNAi or chemical inhibitors to suppress this specific enzyme. The original harmful function is transformed into a targeted suppression strategy that protects heterologous proteins while allowing the plant's general defense responses to remain active and functional.
3Productivity
If heterologous protein expression is increased to improve productivity, then biological output is improved, but the plant's natural defense responses cause more unintended proteolysis
Solution Approach 1:
The patent applies preliminary anti-action by introducing inhibitors of AED3-1 protease before heterologous protein expression begins or at the time of pathogen infection. Through pre-introduction of RNAi constructs or chemical inhibitors, the proteolytic threat is neutralized in advance, allowing high-level heterologous protein expression to proceed without subsequent degradation by the AED3-1 enzyme.
Solution Approach 2:
The patent uses RNAi molecules or chemical inhibitors as intermediary substances that mediate between the heterologous protein expression system and the plant's natural defense responses. These intermediaries specifically target and inhibit AED3-1 protease activity, creating a protective bridge that allows high protein production to occur without triggering harmful proteolytic responses from the plant's immune system.
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 significantly increases the yield and accumulation of heterologous proteins in plants, overcoming the limitations of natural defense responses and proteolysis, thereby improving the efficiency of molecular pharming.
Implementation Method 1
introducing into the plant or plant cell a nucleic acid molecule encoding an inhibitor of aspartic protease AED3-1
Implementation Method 2
introducing into the plant or plant cell a nucleic acid molecule encoding the heterologous protein operably linked to a promoter
Implementation Method 3
growing the plant or plant cell to obtain a plant that expresses the heterologous protein
Implementation Method 4
unintended proteolysis (Grosse-Holz et al., 2018b) that reduce biological output
Data Source
AI summary
The disclosure relates to methods for enhancing production of a heterologous protein in a plant or plant cell by inhibiting aspartic protease AED3-1. Also provided are plants and plant cells with enhanced production of a heterologous protein, wherein the plant or plant cell has reduced or loss of function of AED3-1. Further provided are shRNAs and vectors for use in enhancing production of a heterologous protein in a plant or plant cell.