Fungal Host Cell Selection via Short Intron Riboswitch
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Solution Overview
Problem
The biotech industry faces challenges in developing fungal host cells for high-yield production of polypeptides without relying on antibiotic resistance markers, as existing methods for amplifying expression cassettes in fungal cells are not well established, unlike in Bacillus species.
Innovation Solution
The method involves transforming fungal host cells with a polynucleotide construct containing a spliceosomal intron with 5 nucleotides or less between its branch and acceptor sites, integrated into the genome, and a riboswitch, allowing for adjustable splicing efficiency and low-level expression of selectable markers, enabling multiple copy integration and high-yield production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If antibiotic marker genes are used for selection, then multiple copies of expression cassettes can be amplified efficiently, but the resulting strains contain antibiotic resistance markers which are undesirable for industrial production
Solution Approach 1:
The patent changes the selection mechanism from antibiotic resistance to metabolic auxotrophy complementation. The selectable marker gene (e.g., pyr4 encoding orotidine-5'-phosphate decarboxylase) complements a specific nutritional deficiency (uracil auxotrophy), allowing selection of transformants on media lacking uracil without introducing antibiotic resistance markers.
Solution Approach 2:
The patent uses a transient selectable marker system where the marker gene is integrated alongside the expression cassettes but can be subsequently removed or is naturally lost. The selection pressure (uracil deficiency) is temporary and only needed during the amplification phase, not in final production strains.
2Productivity
If the number of copies of expression cassettes is increased, then product yield increases, but the complexity of achieving stable multi-copy integration without antibiotic markers increases
Solution Approach 1:
The patent employs a dynamic selection strategy where selection pressure is applied during the amplification phase to enrich for multi-copy integrants, then relaxed or modified in production phases. The selectable marker enables flexible transition from selection to production without genetic modification.
Solution Approach 2:
The patent segments the development process into distinct phases: transformation selection using the pyr4 marker, amplification under selection pressure to achieve multi-copy integration, and subsequent production phase where the marker may be removed or the strain used directly. Each phase has optimized conditions for its specific goal.
3Ease of manufacture
If traditional antibiotic selection is used, then the process is simple and well-established, but it is not suitable for fungal host cells unlike Bacillus species
Solution Approach 1:
The patent develops a universal selection system based on metabolic auxotrophy complementation that works across different fungal species (Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei). The pyr4 marker system is broadly applicable to fungi with uracil biosynthesis pathways, providing a versatile platform for industrial fungal host engineering.
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 creation of recombinant fungal host cells with multiple copies of the polynucleotide construct, enhancing product yield by selecting for cells with amplified markers and ensuring high expression levels of the polypeptide of interest.
Implementation Method 1
The thiA riboswithc of A. oryzae contains a nuclear pre-mRNA intron, a spliceosomal intron, which is involved in facilitating the alternative splicing
Implementation Method 2
A riboswitch is part of an mRNA molecule that can bind directly to a small target molecule without a protein being involved. Binding of the small target molecule will affect the translation of the mRNA
Implementation Method 3
a spliceosomal intron which has 5 nucleotides or less between its branch site and its acceptor site... allowing for adjustable splicing efficiency and low-level expression of selectable markers
Data Source
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AI summary
The present invention relates to methods for constructing a recombinant fungal host cell comprising one or more copies of a polynucleotide construct integrated in its genome, said method comprising transforming a fungal host cell with an integrative polynucleotide construct comprising a first polynucleotide encoding a selectable marker, wherein the first polynucleotide, a 5' untranslated region thereof and/or a riboswitch operably linked therewith comprises a spliceosomal intron which has 5 nucleotides or less between its branch site and its acceptor site; and a second polynucleotide encoding a polypeptide of interest; as well as suitable polynucleotide constructs, resulting fungal host cells and methods of manufacture.