RoCi-a single-step multi-copy integration system based on rolling-circle replication.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Fungi are crucial cell factories for producing enzymes and metabolites.
• Generating high-yielding fungal strains often requires integrating multiple gene copies, which is challenging.
• Existing methods for multiple gene integration are often complex and inefficient.

Purpose of the Study:

• To develop a simple and efficient method for integrating multiple gene-expression cassettes into a defined genomic locus in fungi.
• To improve the construction of fungal cell factories for enhanced metabolite and enzyme production.
• To demonstrate the applicability of the method in different fungal species.

Main Methods:

• Developed RoCi (Rolling Circle Integration), a method utilizing rolling-circle replication for gene cassette integration.
• Performed RoCi without the need for Escherichia coli-based cloning, enabling automated experiments.
• Applied RoCi in Aspergillus nidulans to generate strains with up to ~70 copies of mRFP.

Main Results:

• RoCi facilitated the stable integration of multi-copy gene arrays, supporting high and stable mRFP production for at least 150 generations in solid medium.
• A minor copy loss in liquid culture was mitigated by optimizing the gene-expression cassette with a defective promoter-controlled selection marker.
• RoCi application led to a ~10-fold increase in cordycepin and β-glucuronidase production compared to single-copy strains.
• Successfully applied RoCi in industrial fungal workhorses Aspergillus niger and Aspergillus oryzae.

Conclusions:

• RoCi is a simple, efficient, and versatile method for integrating multiple gene copies in fungi.
• The method significantly enhances the production of valuable metabolites and enzymes by fungal cell factories.
• RoCi holds promise for applications in synthetic biology and industrial biotechnology, including in A. niger and A. oryzae.