Abstract
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.
Highlights
Before the advent of DNA-mediated transformation systems in filamentous fungi, random mutagenesis and strain screening programs were the only genetic approaches to strain improvement in biotechnologically relevant fungi
Our findings extend the options for genetic manipulation of this important biotechnological fungus, which is the subject of metabolic engineering experiments, to optimize the production of mycophenolic acid and its analogues [13]
To use bacterial resistance markers for DNA-mediated transformation, we tested the sensitivity of six type culture collection strains from P. brevicompactum against different concentrations of of six type culture collection strains from P. brevicompactum against different concentrations of nourseothricin and phleomycin
Summary
Before the advent of DNA-mediated transformation systems in filamentous fungi, random mutagenesis and strain screening programs were the only genetic approaches to strain improvement in biotechnologically relevant fungi. The previously applied ble [16,17] worked properly in the complementation studies, the commercially available nat could not successfully be used for selection of antibiotic-resistant P. brevicompactum transformants, other studies have applied this resistance marker successfully in filamentous fungi [18,19]. To circumvent this difficulty, we developed a novel codon-adapted nat selection marker that is suitable for high-frequency transformation of P. brevicompactum. Our findings extend the options for genetic manipulation of this important biotechnological fungus, which is the subject of metabolic engineering experiments, to optimize the production of mycophenolic acid and its analogues [13]
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