Aspergillus Chromosomal Deletion via Ku Gene Disruption

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Solution Overview

Problem

Aspergillus strains like Aspergillus sojae and Aspergillus oryzae have a low frequency of homologous recombination, making it difficult to produce strains with large chromosomal deletions, especially for the Aflatoxin biosynthesis gene cluster, which is challenging for food safety and enzyme productivity enhancement.

Innovation Solution

Disrupting the Ku70 and/or Ku80 genes to increase homologous recombination frequency, allowing for the efficient deletion of large chromosomal regions, including the Aflatoxin biosynthesis gene cluster, by integrating homologous regions at both ends of the target chromosomal region using homologous recombination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional methods are used for chromosomal modification in Aspergillus strains, then the process is simple, but the frequency of homologous recombination is very low (1-3%), making it difficult to produce strains with large-area deletions

Engineering Contradiction:

Improvefrequency of homologous recombinationVSAvoiddifficulty of producing deletant strains

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent changes the genetic parameters of the Aspergillus strain by disrupting the ku70 and/or ku80 genes, which are involved in non-homologous recombination. This parameter change increases the frequency of homologous recombination from 1-3% to significantly higher levels, enabling efficient production of strains with large chromosomal deletions including the Aflatoxin biosynthesis gene cluster

Inventive Principle:

Principle #35Parameter changes

2Object-affected harmful factors

If the Aflatoxin biosynthesis gene cluster is to be deleted for food safety, then food safety is improved, but the process is extremely difficult due to low homologous recombination frequency and lack of phenotypic selection markers

Engineering Contradiction:

ImproveAflatoxin production capabilityVSAvoiddifficulty of deleting gene cluster

Core Design Contradiction:

Object-affected harmful factorsVSEase of manufacture

Solution Approach 1:

The patent applies parameter changes by disrupting ku70 and/or ku80 genes to increase homologous recombination frequency, and by introducing phenotypic selection markers (such as antibiotic resistance genes or auxotrophic markers) into the deletion construct, enabling both the deletion of the Aflatoxin biosynthesis gene cluster and the selection of successful transformants

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses intermediary selection markers as mediators to identify and select strains that have successfully integrated the deletion construct. These markers (e.g., antibiotic resistance genes, auxotrophic complementation markers) serve as detectable intermediaries that indicate successful chromosomal modification, allowing researchers to select desired deletant strains from transformants

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If multiple transformants need to be prepared to achieve a single integration, then the desired strain can be obtained, but the time and resources required increase significantly

Engineering Contradiction:

Improvesuccess rate of strain productionVSAvoidtime to prepare and select transformants

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent changes the recombination frequency parameter by disrupting ku70 and/or ku80 genes, which increases the efficiency of homologous recombination. This parameter change reduces the number of transformants that need to be screened and decreases the time required to obtain desired deletant strains, while maintaining high reliability of successful integration

Inventive Principle:

Principle #35Parameter changes

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 enables the efficient production of strains with deleted chromosomal regions, enhancing food safety by eliminating Aflatoxin production capabilities and improving enzyme productivity, overcoming the limitations of low recombination frequencies in Aspergillus strains.

Implementation Method 1

it has been very difficult to produce a strain with a large area-deletion of an arbitrary region in chromosome by conventional methods... as the frequency of homologous recombination is as low as 1~3%... a recent study by the present inventors has revealed that the disruption of a gene involved in non-homologous recombination will significantly increase the frequency of gene targeting (homologous recombination)

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentEP1826273B1Method for the production of a strain having a deleted region in chromosome

Publication Date: 2014.10.15 NODA INST FOR SCI RES

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AI summary

The purpose of the present invention is therefore to provide a transtormani which will not produce a toxic substance such as Aflatoxin even after being manipulated with genetic engineering by efficiently deleting a large chromosomal region with a length of from several tens to hundreds kb such as a cluster of biosynthesis genes encoding the toxic substances such as Aflatoxin. The present invention is related to a method for the production of a strain having a deleted region in chromosome using a transformant having an increased frequency of homologous recombination due to suppression of a Ku gene, which is a mitosporic filamentous fungus belonging to Trichocomaceae, comprising transforming said transformant so as to include a homologous region in both ends of a chromosomal region to be deleted, and deleting the chromosomal region by means of homologous recombination based on said homologous region.