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, 110 (4), 1476-81

Sexual Reproduction and Mating-Type-Mediated Strain Development in the Penicillin-Producing Fungus Penicillium Chrysogenum

Affiliations

Sexual Reproduction and Mating-Type-Mediated Strain Development in the Penicillin-Producing Fungus Penicillium Chrysogenum

Julia Böhm et al. Proc Natl Acad Sci U S A.

Abstract

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1-1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The sexual cycle of P. chrysogenum. (A) Paired culture of Q176 (MAT1-1) and IB 08/921 (MAT1-2) incubated for 5 wk at 20 °C in the dark on oatmeal agar supplemented with biotin. Cleistothecia were found at the junction zones (arrows) of mycelia from both mating types. Scale bar: 2 cm (Top Left). Light (Upper Right) and scanning electron (Middle and Bottom Left and Lower Right) micrographs illustrate cleistothecia (arrows), asci (arrowhead), and ascospores (star), with a chain of smaller conidia for comparison (asterisk). Scale bars: 10 µm (Middle and Bottom Left) and 100 μm (Lower Right). (B) Phenotypic evidence of recombination in selected ascospore progeny (AS), as indicated, compared with parental isolates Q176 and IB 08/921. Front and reverse views show conidial density and chrysogenin formation, respectively. The Bottom row shows results of a penicillin bioassay with halo formation on a bacterial lawn. (C) Molecular evidence of recombination by PCR analysis (Top) and restriction fragment length polymorphism (RFLP) analysis (Middle and Bottom). DNA from four ascospore lineages (designated AS) and two parental strains (Q176 and IB 08/921) was used for analysis with mating-type locus-specific primers. Mating types are distinguished by the size of PCR amplicons. For RFLP analysis, gene-specific primers were used for amplification of DNA, followed by restriction enzyme digestion with PdmI (nsdD) and HinfI (hypo.; Pc24g01940). Strain designation color indicates either MAT1-1 (blue) or MAT1-2 (red) genotype.
Fig. 2.
Fig. 2.
Functional analysis of the MAT1-1–1 gene. (A) Results of a bioassay used to assess penicillin production in parental and representative recombinant MAT1 strains from 48 to 96 h growth. Error bars represent mean ± SD (n = 3) from three independent experiments measuring halo formation. (B) Hyphal morphology of germinating conidia on solid media (24 h, 48 h) and subsequent pellet formation in liquid shaking cultures (72 h, 192 h) formed by parental and representative recombinant MAT1 strains as indicated. Micrographs are representative of three independent experiments. Scale bars: 20 μm (24 h), 100 μm (48 h), and 2,000 μm (72 and 192 h). (C) Quantification of conidia production by parental and representative MAT1 recombinant strains after 168 h growth on complete culture medium in the light or dark. Bottom panel shows typical morphology of respective strains.
Fig. 3.
Fig. 3.
MAT1-1–1 dependent transcriptional regulation. (A) Venn diagram of differentially regulated genes in the ∆MAT1-1–1 EK5 strain. For array analysis, mRNA was used from cultures grown for 36, 60, and 96 h. Arrows indicate transcriptionally up- or down-regulated genes. (B) qRT-PCR analysis to quantify transcriptional expression of the penicillin biosynthesis genes in ΔMAT1-1–1 strains EK5 and EK6 when grown as liquid shaking or surface cultures. Values are mean log2-transformed average expression ratios of at least three biological replicates from two independently derived deletion strains (mean ΔMAT1-1–1 EK5/EK6 n ≥ 3) relative to the ΔPcku70 parental strain.
Fig. 4.
Fig. 4.
Bioassay of functionality of the P. chrysogenum pheromone and pheromone receptor genes. (A) Shmooing of S. cerevisiae in response to the synthetic pheromone PcPPG1 and to S. cerevisiae α-factor. MATa cells (YDB103, ste2Δ, sst2Δ) expressing the Pcpre2 (PcPRE2) gene were treated for 0, 2, or 4 h with either the synthetic pheromone PcPPG1 at 5 µM or DMSO. As a control, the wild type (Wt; MATa strain Y06055 sst2Δ) expressing the endogenous S. cerevisiae STE2 gene was treated with synthetic α-factor or DMSO. On average after 4 h, 50% of cells responded only to the specific pheromone by shmoo formation without any detected cross-reactivity of the pheromones. Scale bar, 10 µm. (B) Pheromone induced growth arrest (halo formation) of S. cerevisiae transformants expressing the P. chrysogenum Pcpre2 gene (PcPRE2; halo diameter: 2.3 ± 0.15 cm), or as a control the S. cerevisiae strain Y06055 (sst2Δ) expressing the endogenous STE2 pheromone receptor gene (ScSTE2p; halo diameter: 2.4 ± 0.13 cm). Averages of halo diameters from eight independent experiments (n = 8) were measured. No cross-reactivity of the pheromones could be detected. DMSO served as mock solution. α, S. cerevisiae α-factor pheromone; Pc, P. chrysogenum decapeptide pheromone (KWCGHIGQGC); Sm, S. macrospora undecapeptide (QWCRIHGQSCW).

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