Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum.

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.

Microarray hybridization analysis of light-dependent gene expression in Penicillium chrysogenum identifies bZIP transcription factor PcAtfA.

The fungal velvet complex is a light-dependent master regulator of secondary metabolism and development in the major penicillin producer, Penicillium chrysogenum. However, the light-dependent mechanism is unclear. To identify velvet-dependent transcriptional regulators that show light-regulated expression, we performed microarray hybridizations with RNA isolated from P. chrysogenum ΔPcku70 cultures grown under 13 different long-term, light-dependent growth conditions. We compared these expression data to data from two velvet complex deletion mutants; one lacked a subunit of the velvet complex (ΔPcvelA), and the other lacked a velvet-associated protein (ΔPclaeA). We sought to identify genes that were up-regulated in light, but down-regulated in ΔPcvelA and ΔPclaeA. We identified 148 co-regulated genes that displayed this regulatory pattern. In silico analyses of the co-regulated genes identified six proteins with fungal-specific transcription factor domains. Among these, we selected the bZIP transcription factor, PcAtfA, for functional characterization in deletion and complementation strains. Our data clearly indicates that PcAtfA governs spore germination. This comparative analysis of different microarray hybridization data sets provided results that may be useful for identifying genes for future functional analyses.

PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum.

Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus.

Among developmental regulators, StuA but not BrlA is essential for penicillin V production in Penicillium chrysogenum.

In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the ß-lactam antibiotic penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA. Inactivation of either brlA or stuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy, but did not affect biomass production during liquid-submerged growth. Genome-wide transcriptional profiling identified a complex StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary metabolism. Remarkably, inactivation of stuA, but not brlA, drastically downregulated expression of the penicillin biosynthetic gene cluster during solid and liquid-submerged growth. In agreement, penicillin V production was wild-type-like in brlA-deficient strains but 99% decreased in stuA-deficient strains during liquid-submerged growth, as shown by high-performance liquid chromatography (HPLC) analysis. Thus, among identified regulators of penicillin V production StuA has the most severe influence. Overexpression of stuA increased the transcript levels of brlA and abaA (another developmental regulator) and derepressed conidiation during liquid-submerged growth but did not affect penicillin V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development and secondary metabolism in P. chrysogenum.

Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum.

Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here, we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, which we have named P. chrysogenum velA (PcvelA) and PclaeA. Data from array analysis using a DeltaPcvelA deletion strain indicate a significant role of PcVelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and high-performance liquid chromatography quantifications of penicillin titers clearly show that both PcVelA and PcLaeA play a major role in penicillin biosynthesis in a producer strain that underwent several rounds of UV mutagenesis during a strain improvement program. Both regulators are further involved in different developmental processes. While PcvelA deletion leads to light-independent conidial formation, dichotomous branching of hyphae, and pellet formation in shaking cultures, a DeltaPclaeA strain shows a severe impairment in conidiophore formation under both light and dark conditions. Bimolecular fluorescence complementation assays provide evidence for a velvet-like complex in P. chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators in genera other than Aspergillus.

The paf gene product modulates asexual development in Penicillium chrysogenum.

Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum.

Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain DeltaPcku70 shows up-regulation of genes from the HOG pathway.

In Penicillium chrysogenum, the industrial producer of the beta-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain DeltaPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain DeltaPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain DeltaPcku70, three genes related to beta-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.

Proper folding of the antifungal protein PAF is required for optimal activity.

The Penicillium chrysogenumantifungal protein PAF is secreted into the supernatant after elimination of a preprosequence. PAF is actively internalized into the hyphae of sensitive molds and provokes growth retardation as well as changes in morphology. Thus far, no information is available on the exact mode of action of PAF, nor on the function of its prosequence in protein activity. Therefore, we sought to investigate the effects of secreted PAF as well as of intracellularly retained pro-PAF and mature PAF on the sensitive ascomycete Aspergillus nidulans, and transformed this model organism by expression vectors containing 5'-sequentially truncated paf-coding sequences under the control of the inducible P. chrysogenum-derived xylanase promoter. Indirect immunofluorescence staining revealed the localization of recombinant PAF predominantly in the hyphal tips of the transformant Xylpaf1 which expressed prepro-PAF, whereas the protein was found to be distributed intracellularly within all segments of hyphae of the transformants Xylpaf2 and Xylpaf3 which expressed pro-PAF and mature PAF, respectively. Growth retardation of Xylpaf1 and Xylpaf3 hyphae was detected by proliferation assays and by light microscopy analysis. Using transmission electron microscopy of ultrathin hyphal sections a marked alteration of the mitochondrial ultrastructure in Xylpaf1 was observed and an elevated amount of carbonylated proteins pointed to severe oxidative stress in this strain. The effects induced by secreted recombinant PAF resembled those evoked by native PAF. The results give evidence that properly folded PAF is a prerequisite for its activity.

Complete Sequencing and Chromosome-Scale Genome Assembly of the Industrial Progenitor Strain P2niaD18 from the Penicillin Producer Penicillium chrysogenum.

Penicillium chrysogenum is the major industrial producer of the ß-lactam antibiotic penicillin. Here, we report the complete genome sequence of the industrial progenitor strain P. chrysogenum P2niaD18 in a chromosome-scale genome assembly. P2niaD18 is distinguished from the recently sequenced P. chrysogenum Wisconsin 54-1255 strain by major chromosomal rearrangements leading to a modified chromosomal architecture.

Genome Sequence and Annotation of Acremonium chrysogenum, Producer of the ß-Lactam Antibiotic Cephalosporin C.

The filamentous fungus Acremonium chrysogenum is the industrial producer of the ß-lactam antibiotic cephalosporin C. Here, we present the genome sequence of strain ATCC 11550, which contains genes for 8,901 proteins, 127 tRNAs, and 22 rRNAs. Genome annotation led to the prediction of 42 gene clusters for secondary metabolites.

A novel homologous dominant selection marker for genetic transformation of Penicillium chrysogenum: overexpression of squalene epoxidase-encoding ergA.

Genetic engineering requires genetic selection markers. For generation of biosafe strains in industrial applications, homologous dominant selection markers allowing "self-cloning" are best suited but scarce. Here we describe a novel homologous dominant genetic selection system for the filamentous fungus Penicillium chrysogenum based on overexpression of the P. chrysogenum squalene epoxidase-encoding ergA gene, which confers resistance against terbinafine. Terbinafine (TRB) is a potent antifungal drug used in therapy of fungal infections. Overexpression of ergA was driven by the P. chrysogenum endoxylanase xylP promoter that is highly inducible by xylose. The suitability of the novel selection marker cassette for genetic manipulation was proven by its use for targeted deletion of the transcription factor nosA in P. chrysogenum. NosA-deficiency did not affect growth rates on solid or in liquid media, conidiation in light or darkness, and resistance to hydrogen peroxide. However, NosA-deficiency significantly decreased penicillin productivity. As TRB inhibits the growth of a variety of fungal species, this novel selection marker is expected to be suitable for genetic engineering of diverse fungal species.

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC).

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.

Regulation of freA, acoA, lysF, and cycA expression by iron availability in Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, iron homeostasis is regulated at the transcriptional level by the negative-acting GATA factor SREA. In this study the expression of a putative heme-containing metalloreductase-encoding gene, freA, was found to be upregulated by iron limitation independently of SREA, demonstrating the existence of an iron-regulatory mechanism which does not involve SREA. In contrast to freA, various other genes encoding proteins in need of iron-containing cofactors-acoA, lysF, and cycA-were downregulated in response to iron depletion. Remarkably, SREA deficiency led to increased expression of acoA, lysF, and cycA under iron-replete growth conditions.