A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger

Area of Science:

• Biotechnology
• Molecular Biology
• Mycology

Background:

• Aspergillus niger is a key industrial fungus for protein production due to its high secretion capacity.
• Existing methods for increasing protein yield often involve random gene integration, which can be inefficient.
• There is a need for precise and efficient methods to increase gene copy number for enhanced protein production in A. niger.

Purpose of the Study:

• To develop a versatile platform for targeted, multicopy gene integration in Aspergillus niger.
• To improve protein production yields by precisely controlling the number and location of integrated genes.
• To demonstrate the utility of this platform for producing specific heterologous proteins.

Main Methods:

• Engineered A. niger strains with up to 10 "glucoamylase landing sites" (GLSs) replacing non-essential genes.
• GLSs contain the promoter and terminator of the highly expressed glucoamylase gene (glaA).
• CRISPR/Cas9 genome editing with unique KORE sequences for targeted integration at specific GLSs.

Main Results:

• Successfully created a system for rapid, targeted gene replacement and integration at multiple genomic sites.
• Demonstrated the ability to generate strains with varying copy numbers of an integrated gene of interest.
• Produced Penicillium expansum PatE::6xHis protein using the platform, achieving approximately 70 μg·mL⁻¹ with high purity (nearly 90%) in a 10-copy strain.

Conclusions:

• The developed GLS platform offers a powerful and precise tool for enhancing heterologous protein production in Aspergillus niger.
• This targeted integration system facilitates rapid strain development and optimization for industrial applications.
• The platform's efficiency was validated by the successful high-yield production of a specific enzyme.