Chimeric Polypeptide Design for Genetic Stability in Transgenic Cells
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Solution Overview
Problem
Current synthetic biology approaches face challenges in maintaining the stability and expression of genetic constructs due to metabolic burdens and random mutations, leading to the loss of target protein expression and instability in microbial populations, which hinders advancements in biosynthetic engineering and therapeutics.
Innovation Solution
A method involving the creation of chimeric polypeptides composed of a polypeptide of interest and an essential protein, optimized through modified GC content, codon usage, and epigenetic modifications, to ensure stable expression in transgenic cells over multiple generations, using a computer program to design and optimize nucleic acid sequences for enhanced genetic stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a foreign gene is expressed in high levels in a microorganism for therapeutics or bioproduction, then the production capacity of the target protein is improved, but the metabolic load on the host increases and genetic stability deteriorates
Solution Approach 1:
The patent combines the foreign target gene with an essential host gene to form a chimeric polypeptide. This merging ensures that the foreign gene cannot be lost or mutated independently, as its expression is coupled with the essential gene required for host survival. The chimeric structure integrates the target protein sequence with the essential protein sequence, creating a single functional unit that maintains both high expression levels and genetic stability.
Solution Approach 2:
The patent performs preliminary optimization of the nucleic acid sequence encoding the chimeric polypeptide before expression. This includes optimizing codon usage to match host preferences, adjusting GC content, and designing the sequence to minimize secondary structures that could interfere with transcription or translation. These preliminary actions ensure high expression capacity while preventing common causes of genetic instability.
2Productivity
If the target gene is expressed at high levels, then the protein production increases, but random mutations occur that reduce or erase target protein expression
Solution Approach 1:
By fusing the target gene with an essential gene, the patent creates a chimeric polypeptide where mutations in the target gene portion would compromise the function of the essential protein portion. This merging protects against random mutations that would otherwise eliminate target protein expression, as such mutations would be selected against due to the essential nature of the combined protein.
Solution Approach 2:
The patent optimizes parameters of the nucleic acid sequence including codon usage frequency to match host cell preferences, GC content to appropriate ranges, and elimination of secondary structures. These parameter changes reduce the likelihood of transcriptional errors, translational errors, and mutational events that could lead to loss of target protein expression.
3Adaptability or versatility
If foreign genes are introduced into microorganisms for biosynthetic engineering, then the functional capabilities are improved, but the evolutionary stability of the genetic construct deteriorates
Solution Approach 1:
The patent merges the foreign biosynthetic gene with an essential host gene to create a chimeric polypeptide. This merging strategy ensures that the foreign gene becomes evolutionarily stable because its retention is coupled with the essential gene required for host survival. The chimeric structure allows the host to maintain the foreign biosynthetic capability while ensuring genetic stability through essential gene coupling.
Solution Approach 2:
The essential gene portion of the chimeric polypeptide serves the host cell's essential functions, while the foreign gene portion provides the desired biosynthetic capability. The essential gene essentially 'pays for' the maintenance of the foreign gene by providing a selective advantage to cells that maintain the chimeric construct, making the system self-sustaining and evolutionarily stable.
Data Source
AI summary
The present invention is directed to a chimeric polypeptide including a polypeptide of interest N-terminal to an essential protein of a target cell. Further provided is a polynucleotide encoding the chimeric polypeptide, a method of producing thereof, and a method for producing a protein of interest. The chimeric polypeptide of the invention improves the genetic stability of the polypeptide of interest ensuring its expression over time.