An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Fungal genetics

Background:

• Metabolic engineering demands efficient genetic tools for rapid development of pathway variants.
• Aspergillus niger is a key industrial fungus, but requires improved genetic manipulation tools.

Purpose of the Study:

• To develop and present a versatile genetic engineering toolbox for Aspergillus niger.
• To enable efficient and flexible generation of strains with heterologous expression cassettes at a defined locus.

Main Methods:

• Utilized Golden Gate cloning for DNA construction and design flexibility.
• Employed a CRISPR/Cas9 strategy for targeted integration into the pyrG locus.
• Used a size-reduced AMA1 variant for transient Cas9 expression and plasmid loss.

Main Results:

• Achieved high integration efficiency, up to 100%, into the fungal genome.
• Significantly reduced the screening process for successful transformants.
• Demonstrated feasibility by integrating an aconitic acid production cassette.

Conclusions:

• The presented toolbox significantly enhances the ease and speed of generating engineered Aspergillus niger strains.
• This system offers a robust and efficient method for constructing complex metabolic pathways in fungi.
• The approach is suitable for various applications, including the production of valuable compounds like aconitic acid.