Polycistronic Expression Construct Using 2A Peptides
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Solution Overview
Problem
Current methods for expressing multiple genes in yeast cells, such as P. pastoris, face challenges with genetic instability due to repeated use of homologous sequences and decreased transformation efficiency with larger expression constructs, making it difficult to implement stable multi-gene pathways for industrial processes.
Innovation Solution
A recombinant polycistronic expression construct using 2A sequences to separate and express multiple genes from a single transcript, avoiding the need for repetitive regulatory elements and enhancing genetic stability and transformation efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple genes are expressed using separate regulatory elements in P. pastoris, then each gene can be independently regulated, but genetic instability occurs due to recombination events from repeated homologous sequences
Solution Approach 1:
The patent merges multiple genes and their regulatory elements into a single polycistronic expression construct. Instead of using separate promoters and terminators for each gene, the invention uses one promoter and one terminator to control the expression of multiple genes arranged in a polycistronic array, thereby eliminating repeated homologous sequences that cause recombination while maintaining independent gene expression through ribosomal skipping at 2A peptide sites.
Solution Approach 2:
The patent segments the polycistronic transcript into individual gene expression units separated by 2A peptide coding sequences. Each gene is divided as an independent functional unit within the polycistronic construct, allowing ribosomal recognition and translation of each gene separately while maintaining the integrity of the entire expression construct and preventing recombination events.
2Productivity
If the expression construct size is increased to include more genes, then more metabolic pathways can be implemented, but transformation rates decrease with increasing construct size
Solution Approach 1:
The patent combines multiple genes into a single polycistronic expression construct that is optimized in size. By sharing common regulatory elements (one promoter and one terminator) across multiple genes and using compact 2A peptide separators, the total construct size is minimized compared to using separate expression cassettes for each gene, thereby maintaining high transformation efficiency while enabling expression of complex multi-gene metabolic pathways.
3Productivity
If traditional IRES sequences are used to couple multiple genes, then coordinate expression is achieved, but nonstoichiometric expression of multiple proteins occurs
Solution Approach 1:
The patent introduces 2A peptide coding sequences as intermediaries between adjacent genes in the polycistronic construct. These 2A peptides mediate ribosomal skipping during translation, causing precise cleavage between the Gly and Pro at the C-terminus of the 2A sequence. This mechanism ensures that each gene product is produced in stoichiometric amounts from the single polycistronic transcript, eliminating the nonstoichiometric expression problem associated with IRES sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The use of 2A sequences allows for stable and efficient expression of up to nine genes, improving genetic stability and transformation efficiency, enabling the implementation of complex metabolic pathways in yeast cells.
Implementation Method 1
They are supposed to cause a ribosome 'skip' resulting in the cleavage of the polycistronic transcript between the Gly and the Pro at the C-terminus of the 2A sequence
Data Source
AI summary
The present invention relates to an optimized metabolic pathway design in P. pastoris. In particular, to a recombinant polycistronic expression construct for stable expression of multiple genes of interest in a yeast cell, preferably in P. pastoris.