A MAT1–2 wild‐type strain from Penicillium chrysogenum: functional mating‐type locus characterization, genome sequencing and mating with an industrial penicillin‐producing strain

Abstract

In heterothallic ascomycetes, mating is controlled by two nonallelic idiomorphs that determine the ‘sex’ of the corresponding strains. We recently discovered mating-type loci and a sexual life cycle in the penicillin-producing fungus, Penicillium chrysogenum. All industrial penicillin production strains worldwide are derived from a MAT1-1 isolate. No MAT1-2 strain has been investigated in detail until now. Here, we provide the first functional analysis of a MAT1-2 locus from a wild-type strain. Similar to MAT1-1, the MAT1-2 locus has functions beyond sexual development. Unlike MAT1-1, the MAT1-2 locus affects germination and surface properties of conidiospores and controls light-dependent asexual sporulation. Mating of the MAT1-2 wild type with a MAT1-1 high penicillin producer generated sexual spores. We determined the genomic sequences of parental and progeny strains using next-generation sequencing and found evidence for genome-wide recombination. SNP calling showed that derived industrial strains had an uneven distribution of point mutations compared with the wild type. We found evidence for meiotic recombination in all chromosomes. Our results point to a strategy combining the use of mating-type genes, genetics, and next-generation sequencing to optimize conventional strain improvement methods.

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

“…In the next step, we applied the Pbnat1 gene containing the vector pPtrpC-Pbnat1 for site-specific deletion of two genes inferred to be involved in fungal development: flbA and MAT1-2-1. flbA encodes a regulator of G-protein signaling that is involved in asexual sporulation and mycelial proliferation, and MAT1-2-1 encodes a transcription factor that controls sexual development [17,34,35]. To generate deletion mutants by homologous recombination, we transformed the strains with a linear deletion cassette containing 5'-and 3'-flanking regions derived from the target gene and each about 1 kb long ( Figures S1A and S2A).…”

“…and MAT1-2-1 encodes a transcription factor that controls sexual development [17,34,35]. To generate deletion mutants by homologous recombination, we transformed the strains with a linear deletion cassette containing 5'-and 3'-flanking regions derived from the target gene and each about 1 kb long ( Figures S1A, S2A).…”

“…Here, we used ble and the nourseothricin-resistance marker nat1 for gene deletion analysis, followed by complementation studies. Although the previously applied ble [16,17] worked properly in the complementation studies, the commercially available nat1 could not successfully be used for selection of antibiotic-resistant P. brevicompactum transformants, although other studies have applied this resistance marker successfully in filamentous fungi [18,19]. To circumvent this difficulty, we developed a novel codon-adapted nat1 selection marker that is suitable for high-frequency transformation of P. brevicompactum.…”

Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains

“…Furthermore, the genetic stability of the isolated strain must be confirmed by various rounds of colony isolation. In contrast to protoplast fusions, sexual recombination of P. chrysogenum strains enables the generation of homokaryotic ascospore isolates, and their increased heat resistance can be used to easily separate ascospores from asexual conidiospores (Böhm et al 2015).…”

“…1) led to increased formation of fruiting bodies containing highly recombinant progeny (Böhm et al 2015). Based on whole genome shotgun sequencing data from parental strains and ascospore isolates, recombination events were identified in the progeny strains (Böhm et al 2015).…”