Aspergillus Chromosome Duplication via Marker-Mediated Translocation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for duplicating and translocating large regions in Aspergillus chromosomes face challenges such as instability due to recombination between homologous sequences, limited culture medium compatibility, and difficulty in removing transformation markers, making it hard to achieve stable and systematic duplication of desired traits.
Innovation Solution
A method involving the integration of transformation marker genes with terminal coding region deficiencies into specific regions of different chromosomes, followed by homologous recombination and double-strand break repair, allowing for the stable translocation of large regions across chromosomes, including those spanning from centromere to telomere, and enabling the recycling of markers for increased copy numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional mutation treatments are used to duplicate chromosome regions, then duplication of large regions can be achieved, but the duplicated regions are unstable due to recombination between homologous sequences
Solution Approach 1:
The patent introduces a selectable marker gene as an intermediary element integrated into the duplicated chromosome region. This marker gene serves as a stabilizing element that prevents recombination between homologous sequences by providing a unique selectable target, thereby maintaining the stability of the duplicated region while allowing for its propagation.
2Ease of operation
If transformation marker genes are integrated into chromosomes for selection, then desired strains can be selected, but the markers cannot be easily removed or recycled
Solution Approach 1:
The selectable marker gene is designed as a separate, modular genetic element that can be independently integrated into the chromosome and subsequently removed. The marker is flanked by specific boundary sequences that allow for its precise excision using restriction enzymes or recombination systems, enabling both selection of transformants and easy removal/recycling of the marker for subsequent experiments.
3Quantity of substance
If large chromosome regions are duplicated in tandem in the same chromosome, then large scale duplication is achieved, but the duplicated sequences are eliminated due to recombination under eutrophic conditions
Solution Approach 1:
The selectable marker gene serves as a protective intermediary integrated within the duplicated region. Its presence creates a genetic barrier that prevents recombination between the duplicated sequences, particularly under eutrophic conditions where selective pressure is relaxed. The marker allows for the maintenance and propagation of the duplicated region without elimination.
4Productivity
If mutation treatments are applied to induce duplication, then strains with useful characteristics can be obtained, but the mutations occur randomly and cannot target specific regions
Solution Approach 1:
The patent employs preliminary action by first integrating a selectable marker gene at a specific chromosomal location before inducing duplication. This pre-positioned marker serves as a template and selection target that guides the duplication process to occur at the desired location, enabling targeted duplication of specific chromosome regions rather than random mutations.
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
This method achieves stable translocation of large regions, such as 1,400 kb, with increased enzymatic activities like protease and α-amylase, and allows for the removal and recycling of markers, ensuring the stability of the duplicated regions even without selective pressure, facilitating efficient breeding of Aspergillus strains for industrial applications.
Implementation Method 1
followed by homologous recombination and double-strand break repair, allowing for the stable translocation of large regions across chromosomes
Data Source
AI summary
The purpose of the present invention is to provide a technique to duplicate and translocate any large region in a chromosome of a fungus belonging to Aspergillus across a wide range, so that it will possible to stably and systematically acquire an Aspergillus strain having a novel trait, which was un-acquirable by conventional techniques. The present invention relates to a transformant of the fungus belonging to Aspergillus wherein a transformation marker gene with deficiency of a terminal part at the 5' or 3' end of its coding region is integrated into an outside of a target region in a chromosome of the fungus subject to duplicated translocation, and a transformation marker gene with deficiency of a terminal part at the 3' or 5' end of its coding region is integrated into an outside of a region in another chromosome of the fungus to be replaced with the target region.