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

Fungi are often used as cell factories for homologous and heterologous production of enzymes and metabolites. To obtain high-yielding strains it is often necessary to insert multiple gene copies and this is not straightforward. We therefore developed RoCi, a simple and efficient method to facilitate integration of multiple directly repeated gene-expression cassettes into a defined genomic locus through rolling-circle replication. RoCi can be performed without need for an Escherichia coli-based cloning step making it convenient for automated experiments. In Aspergillus nidulans, we have used RoCi to generate transformants containing up to ∼70 copies of mRFP and shown that a multi-copy gene array supports high and stable mRFP production for at least ∼150 generations on solid medium. In liquid culture, we observed a minor average copy loss at 1-liter scale. However, this loss was eliminated by extending the gene-expression cassette with a selection marker controlled by a defective promoter. Using RoCi for cell factory construction resulted in a ∼10-fold increase in production of the specialized metabolite cordycepin and the enzyme β-glucuronidase compared to single-copy strains. Finally, we show that RoCi can also be applied in other fungi like the industrial workhorses A. niger and A. oryzae.