Mutant CPT C-Terminal Engineering for High-Molecular-Weight Polyisoprenoids
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Solution Overview
Problem
Conventional techniques struggle to produce high molecular weight polyisoprenoids due to issues with the folding and membrane binding of cis-prenyltransferase (CPT) family proteins, leading to inadequate enzymatic activity.
Innovation Solution
Mutating the C-terminal region of CPT family proteins not found on rubber particles to match the amino acid sequence of those found on rubber particles, allowing for correct folding and deep membrane incorporation, thereby enhancing polyisoprenoid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CPT family protein not found on rubber particles is used directly, then membrane binding ability is poor, but enzymatic activity and polyisoprenoid production are insufficient
Solution Approach 1:
The invention modifies only the C-terminal region of the CPT family protein to match the amino acid sequence of rubber particle-associated CPT, while keeping the N-terminal region unchanged. This localized modification enables membrane binding and correct folding without altering the core enzymatic function, resolving the contradiction between maintaining enzymatic activity and achieving membrane binding capability.
Solution Approach 2:
The invention changes the amino acid sequence parameter of the C-terminal region from the wild-type sequence (not found on rubber particles) to a modified sequence identical or similar to that of rubber particle-associated CPT. This parameter change enables the protein to bind to membranes and achieve correct folding, thereby improving enzymatic activity and polyisoprenoid production.
2Manufacturing precision
If C-terminal region of CPT is mutated to match rubber particle CPT sequence, then correct folding and membrane incorporation are achieved, but this requires precise amino acid sequence modification
Solution Approach 1:
The invention focuses modification only on the C-terminal region of the CPT family protein, leaving the N-terminal region and core enzymatic domains unchanged. This localized approach achieves correct folding and membrane incorporation while minimizing the complexity of genetic modification requirements.
3Productivity
If conventional CPT techniques are used, then production process is simple, but high molecular weight polyisoprenoid production is not achieved
Solution Approach 1:
The invention changes the amino acid sequence parameter of the C-terminal region to enable membrane binding and correct folding, which directly leads to improved enzymatic activity and high molecular weight polyisoprenoid production. This single parameter change resolves the contradiction between production simplicity and productivity.
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 enables the production of higher molecular weight polyisoprenoids, facilitating the creation of high-performance pneumatic tires and rubber products using plant resources in an environmentally friendly manner.
Implementation Method 1
Method for producing polyisoprenoid by reaction catalyzed by mutant cis-prenyltransferase (CPT) family protein
Data Source
AI summary
Provided are a mutant cis-prenyltransferase (CPT) family protein and a method for producing a polyisoprenoid, which enable the production of a high molecular weight polyisoprenoid. Included is a mutant cis-prenyltransferase (CPT) family protein obtained by mutating the amino acid sequence of a C-terminal region of a cis-prenyltransferase (CPT) family protein not found on rubber particles to be identical or similar to the amino acid sequence of a C-terminal region of a cis-prenyltransferase (CPT) family protein found on rubber particles.