Aspergillus Marker Recycling via Loop-Out Region and Dual Selection

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Solution Overview

Problem

Current methods for modifying Aspergillus microorganisms to express desired phenotypes are limited by the lack of recyclable selection marker genes, particularly for marker recycling methods, and the inefficiency of counter-selection systems using existing genes.

Innovation Solution

The development of a transformed Aspergillus microorganism lacking two types of selection marker genes, specifically the pyrG gene and the tryptophan biosynthesis gene, using a nucleic acid fragment with a loop-out region and applying controlled concentrations of 5-FOA and 5-FAA for selection and counter-selection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If marker recycling method is used with pyrG gene as selection marker, then gene replacement can be confirmed easily and selection marker can be reused, but only one type of selection marker gene is available for recycling

Engineering Contradiction:

Improvenumber of recyclable selection marker genesVSAvoidselection pressure strength

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The invention segments the selection marker system into multiple independent recyclable markers (pyrG, trpC, argB genes) that can be used sequentially. Each marker gene is independently deletable and recyclable, allowing multiple rounds of gene replacement without cross-contamination, thus increasing the number of available recyclable markers while maintaining reliable selection pressure for each individual marker.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention implements a systematic process where selection marker genes are temporarily introduced, used for selection, then removed and recovered for future use. The loop-out region mechanism enables precise removal of the marker gene after it has served its selection purpose, allowing the same marker to be discarded and then recovered for subsequent recycling applications, thereby increasing versatility.

Inventive Principle:

Principle #34Discarding and recovering

2Reliability

If counter selection system uses niaD-deficient strain with drug, then selection can be performed, but background growth of non-deficient strains occurs due to weak selection pressure

Engineering Contradiction:

Improveselection pressure strengthVSAvoidbackground growth of non-deficient strains

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The invention converts the previously harmful background growth issue into a beneficial selection mechanism. By using the loop-out region and specific analog substances (5-FOA for pyrG, 5-FAA for trpC, canavanine for argB), the system ensures that only strains with the desired marker deletion can grow, while strains with intact markers are eliminated. This transforms the selection challenge into a reliable counter-selection system that eliminates background growth.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Adaptability or versatility

If multiple selection marker genes are removed from Aspergillus microorganism, then more target genes can be disrupted, but the microorganism loses ability to perform marker recycling

Engineering Contradiction:

Improvenumber of disrupted target genesVSAvoidmarker recycling capability

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The invention creates a universal marker recycling system where multiple different marker genes (pyrG, trpC, argB) can all be used interchangeably for the same purpose of gene replacement. Each marker gene serves multiple functions: initial selection, counter-selection, and recycling. This multi-functionality allows the system to disrupt multiple target genes while maintaining the ability to recycle markers, as each marker can be independently reused after removal.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 approach allows for the efficient and rapid deletion of target genes on the chromosomes of the transformed Aspergillus microorganism, enabling the production of strains with specific phenotypes and facilitating the detection of new phenotypes caused by gene disruptions.

Implementation Method 1

metabolize 5-fluoroorotic acid (5-FOA), which is an analog of orotidine 5'-monophosphate, an intermediate in the biosynthesis of uridine monophosphate, to produce the toxic substance, 5-fluorouracil (5-FU)

Methodology Applied

Scientific EffectMetabolism: Enzyme

Implementation Method 2

replacing a target gene with a selection marker gene by homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS12234466B2<i>Aspergillus </i>microorganism carrying disruptions of multiple genes and a method of producing thereof

Publication Date: 2025.02.25 KIKKOMAN CORP

AI summary

The objective of the present invention is to provide a transformed Aspergillus microorganism lacking at least two types of selection marker genes available for marker recycling method and a composition therefor. The objective can be achieved by a transformed Aspergillus microorganism lacking at least two types of selection marker genes available for marker recycling method on its chromosomes, or a composition for transforming an Aspergillus microorganism containing at least two types of nucleic acid fragments containing a loop-out region and a selection marker gene available for marker recycling method between homologous recombination regions, wherein the selection marker genes contain a tryptophan biosynthesis gene and a gene different from tryptophan biosynthesis gene.