Codon-Optimized FlgM Fusion for Protein Secretion
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Solution Overview
Problem
Current methods for protein production in genetically modified organisms are limited by inefficient translation and secretion processes, leading to suboptimal expression levels and requiring cell lysis for protein recovery, which is inefficient and costly.
Innovation Solution
The use of engineered FlgM coding sequences with synonymous codon changes to modulate translation efficiency and utilize the flagellar Type III secretion system for efficient secretion of proteins into culture media, allowing for increased production and recovery of proteins of interest without cell lysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional protein production methods are used in genetically modified organisms, then protein expression is achieved, but translation efficiency is low and secretion requires cell lysis which is inefficient and costly
Solution Approach 1:
The patent applies parameter changes by systematically optimizing codon usage in the FlgM coding sequence. Specifically, it replaces rare codons with more frequently used synonymous codons to enhance translation efficiency. The patent also modifies amino acid sequences at critical positions (such as the secretion signal region) to improve secretion efficiency through the Type III secretion system, thereby resolving the contradiction between production efficiency and recovery complexity
Solution Approach 2:
The patent introduces an engineered FlgM protein as an intermediary component that facilitates protein secretion through the Type III secretion system. By fusing the protein of interest to engineered FlgM, the system enables efficient secretion without requiring cell lysis, thus simplifying the manufacturing process and reducing complexity while maintaining high productivity
2Productivity
If synonymous codon changes are made to increase translation speed, then protein production increases, but translation accuracy may be compromised
Solution Approach 1:
The patent carefully selects synonymous codon substitutions that optimize translation speed while preserving accuracy. It uses codon usage frequency data to replace rare codons with preferred codons in a controlled manner, focusing on regions where speed enhancement is most beneficial without compromising the reading frame or amino acid sequence integrity. This selective parameter change approach maintains manufacturing precision while improving productivity
Solution Approach 2:
The patent applies local quality by differentiating codon optimization strategies across different regions of the FlgM coding sequence. It focuses codon optimization on specific regions (such as the N-terminal secretion signal and regions with rare codons) while maintaining original codons in regions where accuracy is critical. This localized approach ensures that translation speed is enhanced where needed without compromising overall translation accuracy
3Quantity of substance
If cell lysis is used for protein recovery, then complete protein extraction is achieved, but the process becomes inefficient and costly
Solution Approach 1:
The patent uses engineered FlgM as an intermediary vehicle that enables proteins to be secreted through the Type III secretion system directly into the culture medium. This intermediary approach allows for complete protein recovery without cell lysis, as the secreted proteins can be easily separated from the culture medium through simple centrifugation or filtration, thereby maintaining recovery completeness while dramatically improving production efficiency
Solution Approach 2:
The patent extracts the protein of interest from the cell interior by utilizing the Type III secretion system to transport it directly to the extracellular space. This extraction method occurs during active cell growth rather than requiring post-culture lysis, enabling continuous production and simplifying the recovery process while maintaining high productivity
Data Source
AI summary
The present invention relates to the effects of codon context and synonymous codon changes on mRNA translation and methods of increasing protein production.