Gene Optimization Codon Substitution Polyadenylation Motifs
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Solution Overview
Problem
Transgene proteins in transgenic plants often exhibit low expression due to factors such as weak promoters, aberrant transcript processing, mis-splicing, and poor codon usage compatibility with the host organism, leading to inefficient translation and stability issues.
Innovation Solution
A method for modifying coding sequences of non-plant proteins, such as Bacillus thuringiensis insecticidal proteins, by optimizing codon usage and reintroducing specific polyadenylation motifs to enhance expression levels in plants, involving codon substitution and the strategic introduction of weak polyadenylation motifs to improve transcript stability and translation efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If codon substitution is performed to optimize codon usage for the host organism, then translation efficiency and protein expression levels improve, but the sequence requires significant modification from the original gene
Solution Approach 1:
The patent applies parameter changes by systematically substituting codons in the transgene sequence to match the codon usage frequency of the host organism. This involves changing the nucleotide composition while maintaining the same amino acid sequence, thereby optimizing translation efficiency and protein expression levels in the host organism.
Solution Approach 2:
The patent extracts and removes polyadenylation signal sequences from the transgene coding region that could cause premature transcript processing and reduced expression. By taking out these harmful elements before introducing optimized polyA signals at appropriate locations, the patent prevents aberrant processing while maintaining high expression levels.
2Reliability
If polyadenylation signal sequences are present in the transgene, then transcript processing may occur, but premature polyadenylation leads to reduced expression and transcript instability
Solution Approach 1:
The patent converts the potentially harmful effect of polyadenylation signals within the coding region by strategically placing optimized polyA signal sequences at specific locations downstream of the coding region. This ensures that polyadenylation occurs only at the correct position, preventing premature processing while maintaining transcript stability and high expression levels.
Solution Approach 2:
The patent performs preliminary removal of problematic polyadenylation signal sequences from the coding region during the gene optimization process, before transformation into the host organism. This preliminary action prevents premature polyadenylation and ensures that only correctly positioned polyA signals remain to facilitate proper transcript processing.
3Adaptability or versatility
If transgenes are derived from other plant species or non-plants, then genetic diversity is achieved, but codon usage differences lead to poor translation efficiency in the host organism
Solution Approach 1:
The patent applies parameter changes by analyzing the codon usage frequency of the host organism and systematically substituting codons in the transgene sequence to match these preferences. This maintains the ability to use diverse gene sources while optimizing translation efficiency for the specific host organism through codon adaptation.
4Productivity
If weak promoters are used to drive transgene expression, then gene expression can be maintained at low levels, but protein expression remains insufficient for practical applications
Solution Approach 1:
The patent optimizes multiple parameters including promoter selection, codon usage frequency, and polyadenylation signal positioning to collectively enhance protein expression levels. By changing these parameters simultaneously, the patent achieves high expression levels without requiring excessively strong promoters that might cause other issues.
Data Source
AI summary
The invention relates to method of modifying a coding sequence encoding a non-plant protein, comprising the steps of optimizing said coding sequence by codon substitution, thereby obtaining an optimized coding sequence which encodes said non-plant protein; and re-introducing at least one wild-type polyadenylation motif sequence at its position within said optimized gene sequence.