Shotgun proteomics reveals putative polyesterases in the secretome of the rock-inhabiting fungus Knufia chersonesos

Abstract

Knufia chersonesos is an ascomycotal representative of black fungi, a morphological group of polyextremotolerant melanotic fungi, whose ability to resort to recalcitrant carbon sources makes it an interesting candidate for degradation purposes. A secretome screening towards polyesterases was carried out for the fungus and its non-melanized mutant, grown in presence of the synthetic copolyester Polybutylene adipate terephthalate (PBAT) as additional or sole carbon source, and resulted in the identification of 37 esterolytic and lipolytic enzymes across the established cultivation conditions. Quantitative proteomics allowed to unveil 9 proteins being constitutively expressed at all conditions and 7 which were instead detected as up-regulated by PBAT exposure. Protein functional analysis and structure prediction indicated similarity of these enzymes to microbial polyesterases of known biotechnological use such as MHETase from Ideonella sakaiensis and CalA from Candida antarctica. For both strains, PBAT hydrolysis was recorded at all cultivation conditions and primarily the corresponding monomers were released, which suggests degradation to the polymer’s smallest building block. The work presented here aims to demonstrate how investigations of the secretome can provide new insights into the eco-physiology of polymer degrading fungi and ultimately aid the identification of novel enzymes with potential application in polymer processing, recycling and degradation.

Effects of Simulated Microgravity on the Proteome and Secretome of the Polyextremotolerant Black Fungus Knufia chersonesos

“…One further interesting aspect was the high number of proteins traditionally recognized as cytoplasmic in K. chersonesos secretome, especially in the mutant (i.e., 6% of total secreted proteins with predicted signal peptide v/s 25% in the wild type; 20% of regulated secreted proteins had signal peptides in both strains). Such a discrepancy between wild type and mutant was also reported in a study of K. chersonesos secretome aimed at the screening for novel polyesterases ( Tesei et al, 2020 ), hence it seems to suggest that a low number of extracellular proteins may be a peculiarity of the mutant secretome. The presence of cytoplasmic proteins in the culture supernatant may indicate that the mutant is more prone to cell lysis than the wild type ( Miura and Ueda, 2018 ).…”

“…Also in the wild type, the casein kinase I 1 CF317_004827-T1 and the histone H2A CF317_001633-T1, both involved in DNA repair ( Skoneczna et al, 2018 ), were found in the LSSMG-exposed secretome at levels 1.7- and 1.6-fold higher than in the unexposed one. The two downregulated proteins were protein CF317_008131-T1, homolog of the arabinan endo-1,5-alpha-L-arabinosidase from Aspergillus wentii DTO 134E9 , and protein CF317_002949-T1, homolog of the feruloyl esterase A0A177BY00_9PLEO from Paraphaeosphaeria sporulosa , both with a predicted extracellular localization and reportedly involved in polysaccharide degradation ( Tesei et al, 2020 ).…”

“…K. petricola ) is an emerging model organism for analyses aiming at the elucidation of the rock-lifestyle and RIF physiology ( Nai et al, 2013 ). Along with its ascertained thermo-, pH-, UV- and desiccation tolerance ( Gorbushina et al, 2008 ; Sterflinger et al, 2012 ), the fungus was recently reported to have an aptitude for using synthetic polymers as an alternative carbon source ( Tesei et al, 2020 ). These features and the ability to endure high levels of gaseous ozone (Tesei et al, unpublished), altogether make K. chersonesos particularly suited for astrobiology studies.…”

“…Information about the predicted protein localization was obtained using BUSCA 3 , based on the identification of signal and transit peptides, GPI-anchors and alpha-helical and beta-stranded transmembrane domains ( Savojardo et al, 2018 ). Protein-protein interaction analyses were performed using STRING v11.0 with high confidence (0.70) ( Jensen et al, 2009 ), selecting the proteome of the black yeast Exophiala dermatitidis as reference database based on its phylogenetic proximity to K. chersonesos ( Tesei et al, 2020 ).…”

“…The rock-associated Knufia chersonesos (syn. Knufia petricola ) was selected as the model organism due to its reported poikilo-tolerance, e.g., the ability to endure xeric conditions, desiccation, high UV-radiation and temperatures ( Sterflinger et al, 2012 ) and to feed on alternative carbon sources like monoaromatic compounds ( Nai et al, 2013 ) and synthetic polyesters ( Tesei et al, 2020 ). K. chersonesos aptitude to withstand levels of gaseous ozone up to 11 ppm was also shown (Tesei et al, unpublished).…”