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Nutrient Excess Triggers the Expression of the Penicillium chrysogenum Antifungal Protein PAFB.

ABSTRACT: Nutrient limitation and nonfavorable growth conditions have been suggested to be major triggers for the expression of small, cysteine-rich antimicrobial proteins (AMPs) of fungal origin, e.g., the Penicillium chrysogenum antifungal protein (PAF), the Aspergillus giganteus antifungal protein (AFP), the Aspergillus niger antifungal protein (AnAFP). Therefore, these AMPs have been considered to be fungal secondary metabolite products. In contrast, the present study revealed that the expression of the PAF-related AMP P. chrysogenum antifungal protein B (PAFB) is strongly induced under nutrient excess during the logarithmic growth phase, whereas PAFB remained under the detection level in the supernatant of cultures grown under nutrient limitation. The efficiency of the pafB-promoter to induce PAFB expression was compared with that of two P. chrysogenum promoters that are well established for recombinant protein production: the paf-promoter and the xylose-inducible promoter of the xylanase gene, xylP. The inducibility of the pafB-promoter was superior to that of the xylP-promoter yielding comparable PAFB amounts as under the regulation of the paf-promoter. We conclude that (i) differences in the expression regulation of AMPs suggest distinct functional roles in the producer beyond their antifungal activity; and (ii) the pafB-promoter is a promising tool for recombinant protein production in P. chrysogenum, as it guarantees strong gene expression with the advantage of inducibility.

SUBMITTER: Huber A

PROVIDER: S-EPMC6956099 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Nutrient Excess Triggers the Expression of the Penicillium chrysogenum Antifungal Protein PAFB.

Huber Anna A Lerchster Hannah H Marx Florentine F

Microorganisms 20191204 12

Nutrient limitation and nonfavorable growth conditions have been suggested to be major triggers for the expression of small, cysteine-rich antimicrobial proteins (AMPs) of fungal origin, e.g., the Penicillium chrysogenum antifungal protein (PAF), the Aspergillus giganteus antifungal protein (AFP), the Aspergillus niger antifungal protein (AnAFP). Therefore, these AMPs have been considered to be fungal secondary metabolite products. In contrast, the present study revealed tha ...[more]

PMID: 31817241

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Project description:BACKGROUND:Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS:The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ?paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION:This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

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Nutrient Excess Triggers the Expression of the Penicillium chrysogenum Antifungal Protein PAFB.

Publications

Nutrient Excess Triggers the Expression of the <i>Penicillium chrysogenum</i> Antifungal Protein PAFB.

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