Optimizing BoNT/E Expression via Codon and GC Content
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Solution Overview
Problem
Current methods for producing Botulinum neurotoxins (BoNTs) like BoNT/A and BoNT/E in heterologous organisms are inefficient, leading to suboptimal yields and higher production costs, especially for toxins that require in vitro activation, such as BoNT/E, due to losses during the activation process.
Innovation Solution
The use of modified nucleic acid molecules with optimized codon usage, G+C content, and reduced polymononucleotide regions to enhance expression levels in heterologous cells, including prokaryotic and eukaryotic systems, to achieve higher yields of active BoNTs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional heterologous expression methods are used to produce BoNTs, then production cost is reduced, but productivity and yield are insufficient
Solution Approach 1:
The patent optimizes nucleic acid parameters including codon usage to match host organism preferences, adjusts G+C content to optimal ranges, and modifies polymononucleotide regions to eliminate secondary structures that inhibit translation. These parameter changes significantly enhance protein expression levels and production yield of BoNTs in heterologous systems
Solution Approach 2:
The patent creates optimized copies of the BoNT/E coding sequence by synthesizing modified nucleic acid molecules that replicate the original gene's function but with improved expression characteristics. These synthetic copies incorporate codon optimization and structural modifications to achieve high-level production
2Reliability
If in vitro activation is used for BoNT/E, then active toxin is produced, but substance loss occurs during activation
Solution Approach 1:
The patent performs preliminary optimization of the coding sequence before expression, incorporating codon optimization and structural modifications that enhance translation efficiency and protein stability. This preliminary action ensures maximum yield of active toxin while minimizing losses during subsequent activation procedures
3Productivity
If codon optimization is applied to nucleic acid molecules, then protein expression level increases, but nucleic acid sequence complexity increases
Solution Approach 1:
The patent systematically modifies nucleic acid parameters including codon composition, G+C content, and polymononucleotide distribution to optimize expression while maintaining sequence manageability. These controlled parameter changes achieve high protein production without excessive sequence complexity
Data Source
AI summary
Nucleic acid molecules that comprise modified open reading frames providing increased expression of the encoded active BoNT/E in a heterologous cell, expression constructs and cells comprising such nucleic acid molecules and methods useful for expressing the encoding active BoNT/E from such nucleic acid molecules, expression constructs and cells.