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9th Trends in Medical Mycology Held on 11–14 October 2019, Nice, France, Organized under the Auspices of EORTC-IDG and ECMM
Open AccessArticle

Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum

Allgemeine & Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany
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Author to whom correspondence should be addressed.
Present address: BASF SE, Carl-Bosch Straße 38, 67056 Ludwigshafen am Rhein, Germany.
J. Fungi 2019, 5(4), 96; https://doi.org/10.3390/jof5040096
Received: 3 September 2019 / Revised: 1 October 2019 / Accepted: 6 October 2019 / Published: 10 October 2019
Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum. View Full-Text
Keywords: Penicillium brevicompactum; codon-adapted nourseothricin resistance gene; homologous recombination; MAT1-2-1; flbA Penicillium brevicompactum; codon-adapted nourseothricin resistance gene; homologous recombination; MAT1-2-1; flbA
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Mahmoudjanlou, Y.; Hoff, B.; Kück, U. Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum. J. Fungi 2019, 5, 96.

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