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Production and biotechnological use of new anti-fungal proteins from filamentary fungi

Thesis

<http://hdl.handle.net/10251/113162>

Abstract

Antimicrobial peptides (AMP) are a promising alternative for the development of new antifungics that can replace fungicides used in agriculture. However, the high cost of chemical synthesis and the difficulty of producing it on a large scale have limited its implementation. Antifungal proteins (AFP) are natural, small, cationic, secreted and cystein-rich AMPs with high potential for the control of plant pathogens. AfPS are found in filamentary fungi, stable and can be produced in large quantities. However, the biological paper in its producer fungus is not fully known. In this thesis, the diversity of AfPS in genomes of ascomicete fungi was studied and a new classification into three classes (A, B and C) was proposed. Penicillium digitatum is the main post-harvest citrus pathogen and encodes only one AFP in its Class B genome (AfpB), while Penicillium expansum, the main post-harvest apple pathogen, encodes one AFP of each class (AFPA, AfpB and AFPC). In this work we describe the biotechnological production and characterisation of these four AfPS. The biological role of the afpB gene in P. digitatum has been characterised by studies of gene expression and generation of zero mutants and constitutive expression. The results indicated that afpB is not necessary for fungus biology and life cycle, although the expression of the afpB gene under the constitutive promoter gpdA of Aspergillus nidulans is detrimental to its growth and virulence. Surprisingly, neither the parental strain nor the constituent strains produced detectable amounts of AfpB despite the high expression of the coding gene. Molecular modelling and rational design allowed predicting AfpB’s tertiary structure and designing synthetic peptides to map antifungal motifs in its primary sequence. We confirm that the cationic loops L2 and L3 showed moderate antifungal activity and that they can act synergetically. With the aim of producing AfpB by means of biotechnology, we use an AfPS expression cassette based on the regions promoting and finishing the PAF gene of Penicillium chrysogenum, a fungus that naturally produces large quantities of its own PAF protein. This cassette operated in P. digitatum and enabled AfpB’s homologous production. The data also showed that the sequences of the signal peptide (SP) and the SP-Pro-AfpB pro-peptide do not determine protein production. We also demonstrate the thermal stability and proteolysis resistance of AfpB and provide data suggesting that tertiary structure is not necessary for antifungal activity. Similar to that described in P. digitatum, none of the three AfPS was detected in the growing supernatants in a rich P. expansum medium. However, AFPA was produced in large quantities in crops with a minimum medium of P. expansum. To complete the AfPS directory, we produced P. expansum’s three AfPS (AFPA, AfpB and AFPC) in P. chrysogenum with the PAF cassette. P. expansum’s three proteins were produced, purified and characterised successfully. None of the AfPS produced in this work was cytotoxic against mammalian erythrocytes. AFPA of P. expansum followed by P. digitatum’s AfpB were the most active AfPS against filamentary fungi, including plant and human pathogens, mycotoxin producers and their own producer fungi, a characteristic not previously described in the AfPS. In addition, P. expansum’s AFPA and P. digitatum’s AfpB protected against fungus Botrytis cinerea infection in tomato plants, and P. expansum’s AFPA protected P. digitatum in orange fruits. These results confirm our assumption that AfPS are good candidates for the development of new antifungics in plant protection and post-harvest conservation, but Garrigues Cubells, SM. (2018). Biotechnological production and use of new anti-fungal proteins from filamentary fungi [Tesis doctoral not published]. Universitat Politèca de València. https://doi.org/10.4995/Thesis/10251/113162

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