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

Abstract Fungi are often used as cell factories for homologous and heterologous production of enzymes and metabolites. One strategy to obtain high yielding strains is to enhance the expression level of the gene(s) responsible for production of the product by inserting multiple copies of the gene-expression cassette. Typically, this is achieved by transforming non-homologous end-joining proficient strains with large amounts of a DNA vector, which randomly integrates in multiple copies at different loci, or more often, into a single locus with copies arranged as mixed orientation repeats. The majority of strains produced in this manner are unstable and substantial screening is necessary to identify strains with high and stable production. Moreover, the randomness of the insertion processes makes it difficult to determine how and where the copies are positioned in the genome. To this end, we envisioned that the instability of gene clusters made by the classical method is mostly due to the presence of a mixture of directly and inverted repeats. In such clusters, hairpins formed by inverted repeats may cause frequent recombinogenic lesions during replication to induce gene-expression cassette copy-loss by direct-repeat recombination. It is therefore possible that strains with gene-expression cassette clusters made solely by direct repeats would be more stable. Using Aspergillus nidulans as a model, we tested this idea and 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 without pre-engineering requirements for strain preparation. In addition, we demonstrate that RoCi can be performed without E. coli based cloning, making it compatible with medium-high throughput experiments. Analyzing strains produced by RoCi, we have constructed strains bearing up to 68 mRFP GECs and we show that an mRFP 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 L scale. This loss could be eliminated by extending the gene-expression cassette with a crippled selection marker. To demonstrate the strength of the method, we used it to produce stable and high yielding cell factories for production of the specialized metabolite cordycepin on solid medium and of the enzyme β-glucuronidase in submerged culture. Finally, we show that RoCi can also be applied in the industrial workhorses A. niger and A. oryzae indicating that RoCi is generally applicable in fungi.