Multicopy Gene Insertion in Trichoderma reesei via TEF1 Promoter
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Solution Overview
Problem
Current methods for inserting genes into the genome of Trichoderma reesei fungus are inefficient, requiring large flanking sequences and having a low probability of precise insertion, which limits the expression of proteins of interest.
Innovation Solution
A method using a vector with a TEF1 promoter identical to the endogenous TEF1 promoter and a terminator different from the native TEF1 terminator, allowing for multicopy insertion of a gene of interest, with the option of using a single flanking region for targeting the TEF1 locus, enhancing protein production capacity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional gene insertion methods are used in Trichoderma reesei, then transformation can be achieved, but the insertion probability is low and requires large flanking sequences
Solution Approach 1:
The patent changes the target insertion locus from conventional sites to the TEF1 promoter region, and modifies the expression cassette design by using the native TEF1 promoter with a heterologous terminator. This parameter change enables efficient multicopy insertion without requiring large flanking sequences, directly resolving the contradiction between insertion precision and device complexity
Solution Approach 2:
The patent employs multiple copies of the expression cassette containing the gene of interest inserted into the TEF1 promoter region. This copying strategy increases the expression level of the target gene while using a streamlined vector design without large flanking sequences, thus achieving both high insertion efficiency and reduced complexity
2Productivity
If single copy gene insertion is used, then genome stability is maintained, but protein production capacity is limited
Solution Approach 1:
The patent deliberately introduces multiple copies of the expression cassette into the TEF1 locus, creating a multicopy arrangement that amplifies protein production capacity. The use of the essential TEF1 promoter region as the insertion target ensures that these multiple copies are stably maintained in the genome, resolving the contradiction between productivity and stability
3Adaptability or versatility
If heterologous promoter is used for gene expression, then expression flexibility is improved, but endogenous regulation compatibility is lost
Solution Approach 1:
The patent uses the native TEF1 promoter, which is a constitutively active promoter in Trichoderma reesei, to drive expression of the gene of interest. This approach maintains compatibility with endogenous regulatory mechanisms while providing reliable expression. The heterologous terminator from Aspergillus nidulans adds functionality by providing efficient transcription termination, achieving both universality and reliability
Data Source
AI summary
The invention relates to a method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus by using a vector comprising an expression cassette, said cassette comprising: (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the species of fungus, (2) a gene of interest, and (3) a terminator that is not the endogenous TEF1 terminator of the fungus species. The invention also relates to the fungi obtained by such a method, and to the various uses of the strains modified in this manner.(no figure).