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RoCi-a single-step multi-copy integration system based on rolling-circle replication
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DOI：10.1093/nar/gkaf1488.png)
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Abstract
En 中文
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 similar to 70 copies of mRFP and shown that a multi-copy gene array supports high and stable mRFP production for at least similar to 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 similar to 10-fold increase in production of the specialized metabolite cordycepin and the enzyme beta-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.
Keywords:
ASPERGILLUS-NIDULANS
FILAMENTOUS FUNGI
GENE-EXPRESSION
SACCHAROMYCES-CEREVISIAE
NUMBER INTEGRATION
DUPLICATION CYCLE
PROTEIN-SYNTHESIS
DNA-REPLICATION
DELTA SEQUENCES
RIBOSOMAL DNA
Journal
IF:
13.1
Papers:
3.6W
Citations:
29.0W