Ku Gene Suppression in Aspergillus for Homologous Recombination
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Solution Overview
Problem
The low frequency of homologous recombination in Aspergillus sojae and Aspergillus oryzae strains complicates genetic analysis and gene disruption, making it difficult to produce desired gene-disruption strains due to the lack of efficient genetic tools and high drug resistance, which hinders industrial applications such as brewed food production.
Innovation Solution
A transformant with increased homologous recombination frequency is achieved by suppressing the ku gene through gene disruption or antisense RNA methods, specifically in Aspergillus sojae and Aspergillus oryzae, leading to a significant enhancement in gene targeting efficiency without negative phenotypic effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ku gene is suppressed to increase homologous recombination frequency, then gene targeting efficiency is improved, but non-homologous recombination capability may be affected
Solution Approach 1:
The patent changes the functional state of the ku70 and ku80 genes from active to suppressed (via disruption or antisense RNA), thereby altering the recombination mechanism balance. This parameter change shifts the system from equal utilization of both recombination pathways to predominant homologous recombination, achieving high gene targeting efficiency while maintaining genomic stability through controlled suppression rather than complete elimination of ku gene function
2Ease of manufacture
If conventional transformation methods are used in Aspergillus sojae and Aspergillus oryzae, then drug resistance selection is simple, but homologous recombination frequency is low
Solution Approach 1:
The patent extracts and suppresses the ku70 and ku80 genes that are responsible for non-homologous recombination. By removing the interference of these genes, the patent enables homologous recombination to dominate the transformation process, thereby dramatically increasing the frequency of desired gene targeting events while maintaining the simplicity of drug resistance selection methods
3Productivity
If gene disruption methods are used to suppress ku70 and ku80, then homologous recombination frequency increases significantly, but genetic tool complexity increases
Solution Approach 1:
The patent performs preliminary suppression of ku70 and ku80 genes before introducing the target gene construct. This preliminary action creates a permissive genetic environment where homologous recombination is highly efficient, allowing subsequent transformation operations to proceed with high success rates without requiring complex screening or selection protocols
Data Source
AI summary
The purpose of the present invention is to provide a strain having a significantly increased frequency of homologous recombination, which is necessary for gene disruption of gene replacement by gene targeting of a mitosporic filamentous fungus. This invention relates to a transformant having an increased frequency of homologous recombination (gene targeting), which is a mitosporic filamentous fungus belonging to Trichocomaceae, due to suppression of a ku gene by ku gene disruption or antisense RNA method, to a method for the production of a gene-disruption stain, gene-deletion strain, gene-replacement strain, gene-insertion strain or chromosome-modification strain by means of the gene targeting method using said transformant, and to the above-mentioned ku genes such as those derived from Aspergillus sojae and Aspergillus oryzae, and their expressed products (proteins).