tRNA Gene Integration into rRNA Operons for Protein Expression
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Solution Overview
Problem
Bacterial host cells face challenges in expressing proteins due to the use of rare codons, leading to low levels of tRNA depletion and requiring inefficient processes like multiple steps of mutagenesis or plasmid introduction, which are costly and unstable.
Innovation Solution
Introducing one or more tRNA genes into the rRNA operon of host cells, chosen based on codon frequency, to increase tRNA levels and enhance protein expression efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If rare codons are used in the gene being expressed, then the natural codon usage of the host cell is maintained, but protein expression is significantly reduced due to depletion of rare tRNAs
Solution Approach 1:
The patent introduces tRNA genes into the host cell genome before protein expression occurs, so that the rare tRNAs are pre-supplied in the cell. This preliminary action ensures that when the gene with rare codons is expressed, the necessary tRNAs are already available, preventing depletion and maintaining high expression levels.
Solution Approach 2:
The patent uses tRNA genes as intermediary elements that mediate between the rare codons in the target gene and the host cell's translation machinery. By introducing these tRNA genes, the cell can translate rare codons efficiently without requiring changes to the original gene sequence or the host's native tRNA pool.
2Productivity
If multiple steps of mutagenesis are performed to replace rare codons with abundant codons, then protein expression is improved, but the process becomes complex and time-consuming
Solution Approach 1:
Instead of modifying the gene sequence to replace rare codons with abundant codons (the conventional approach), the patent inverts the strategy by modifying the tRNA pool rather than the gene. This inversion simplifies the process from multiple mutagenesis steps to a single introduction of tRNA genes, reducing complexity while maintaining expression improvement.
Solution Approach 2:
The patent changes the parameter of tRNA availability by introducing additional tRNA genes into the host cell genome. This parameter change increases the concentration of rare tRNAs, allowing the original gene with rare codons to be expressed efficiently without any changes to the gene sequence itself.
3Productivity
If plasmids containing rare tRNA genes are introduced into host cells, then protein expression is improved, but production costs increase due to selectable markers and genetic instability
Solution Approach 1:
The patent extracts the unnecessary plasmid载体 and selectable marker components from the system by directly integrating tRNA genes into the host cell's chromosomal genome. This extraction eliminates the need for plasmid maintenance, reduces genetic instability, and removes the cost of selectable markers while maintaining the ability to supply rare tRNAs.
Solution Approach 2:
The patent merges the tRNA gene introduction with the host cell's chromosomal genome rather than using separate plasmid vectors. This merging integrates the tRNA supply mechanism into the cell's permanent genetic structure, eliminating the need for additional plasmid-based systems and reducing overall system complexity and cost.
Data Source
AI summary
The invention relates to host cells with improved protein expressed properties. The host cells comprise rare tRNA genes within the one or more rRNA operons.