rDNA NTS Gene Insertion Cassette for Markerless Yeast Engineering
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Solution Overview
Problem
Current methods for inserting genes into yeast chromosomes rely on antibiotic-resistant markers, which pose safety and environmental concerns, and do not allow for efficient multiple insertions without disrupting gene expression stability.
Innovation Solution
A gene multiple insertion cassette set using N-terminal and C-terminal fragments of Saccharomyces cerevisiae ribosomal DNA nontranscribed spacer (rDNA NTS) with an auxotrophic selection marker, enabling multiple insertions into yeast chromosomes without antibiotic-resistant markers, thereby facilitating stable expression of target genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If antibiotic-resistant markers are used for gene insertion into yeast chromosomes, then gene insertion efficiency is improved, but safety and environmental concerns worsen
Solution Approach 1:
The patent extracts and removes the antibiotic-resistant marker component from the gene insertion system. Instead of using traditional antibiotic resistance genes (such as ampicillin resistance), the invention employs a markerless homology-directed repair system where linear DNA fragments with homology arms directly mediate gene insertion into the yeast genome without requiring any antibiotic selection markers, thereby eliminating the safety and environmental hazards associated with antibiotic resistance.
Solution Approach 2:
The patent introduces linear DNA fragments with homology arms as intermediary molecules that facilitate gene insertion without requiring antibiotic markers. These linear fragments serve as mediators that provide the necessary homology for precise genomic integration through homology-directed repair, replacing the traditional role of antibiotic-resistant plasmids and eliminating the need for antibiotic selection.
2Quantity of substance
If multiple genes are inserted into yeast chromosomes using traditional methods, then gene expression stability deteriorates, but gene insertion capacity is improved
Solution Approach 1:
The patent segments the gene insertion process into modular linear DNA fragments, each containing specific homology arms that target particular genomic loci. This segmentation allows multiple genes to be inserted at different locations in the yeast genome simultaneously or sequentially, with each insertion event being independent and precise, thereby maintaining gene expression stability even when multiple genes are introduced.
Solution Approach 2:
The patent applies local quality by designing linear DNA fragments with specific homology arm sequences that are tailored to match particular genomic regions. Each linear fragment possesses localized homology characteristics that ensure precise integration at the intended site while leaving other parts of the genome unaffected, thus maintaining overall genomic stability and normal gene expression patterns despite multiple insertions.
3Ease of operation
If antibiotic-resistant markers are used for selection, then selection efficiency is improved, but environmental safety worsens
Solution Approach 1:
The patent extracts and eliminates antibiotic-resistant markers from the selection process entirely. Instead of relying on antibiotic resistance for selecting successful transformants, the invention uses a markerless homology-directed repair system where selection can be performed through phenotypic complementation or other non-antibiotic methods, thereby removing the source of environmental harm while maintaining practical selection efficiency.
Data Source
AI summary
Disclosed is a gene multiple insertion cassette set including rDNA NTS fragments and an auxotrophic selection marker having an incomplete promoter is developed, and a safe oral recombinant strain having no antibiotic resistant marker is constructed by multiple insertion of an optimum number of the developed gene multiple insertion cassette sets into chromosomes of a Saccharomyces cerevisiae strain, a vaccine composition including, as an active ingredient, the above strain, a culture product thereof, a cell lysate, or nodavirus capsid protein (NNVcp) isolated and purified therefrom, and a composition for feed addition including, as an active ingredient, the above strain, a culture product thereof, a cell lysate, or squalene or oxidosqualene isolated and purified therefrom.