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\(\textit {Penicillium brevicompactum}\) 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 \(\textit {P. brevicompactum}\). Although a generally available phleomycin resistance marker \(\it (ble)\) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette \(\textit {(nat1)}\). To circumvent this failure, we constructed a new nat gene, considering the codon bias for \(\textit {P. brevicompactum}\). We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, \(\textit {MAT1-2-1}\) and \(\it flbA\). For \(\textit {MAT1-2-1}\), 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 \(\it ble\) to reintroduce the wild-type genes. This step restored the wild-type phenotype in the \(\it flbA\) deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus \(\textit {P. brevicompactum}\).

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases.