Open Access
Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus
(Nature Research, 2020) Alfaro Sánchez, Manuel; Majcherczyk, A.; Kües, Ursula; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain.
Open Access
Genomic, transcriptomic and proteomic analysis of Pleurotus ostreatus secreted proteins
(2017) Alfaro Sánchez, Manuel; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
The objective of this thesis is to study the proteins secreted by the edible and worldwide cultivated white rot basidiomycete fungus Pleurotus ostreatus with three major goals: to determine the set of proteins secreted under different nutritional conditions, to determine the effect of the monokaryotic and dikaryotic mycelial conditions on the secretome, and to explore the relationship between the transcriptome of the secreted proteins and the actual secretome in different monokaryotic strains. In the first chapter of this thesis, we will review several basidiomycete secretome analyses comparing the results obtained using different analytical techniques and discussing some representative examples. We will pay a special attention to the lignocellulolytic enzymes secreted and to the different fungal lifestyles. This chapter is an updated version of the paper entitled Comparative analysis of secretomes in basidiomycete fungi that we published in Journal of Proteomics in 2014 as a summary of the state of the art. The main conclusions of this chapter are that a combination of genomic, transcriptomic and proteomics techniques is still the best approach for analyzing fungal secretomes, allowing to the identification of secretion patterns associated to the different lifestyles. In the third chapter, we screened two P. ostreatus monokaryotic genomes to identify bioinformatically the genes coding for proteins targeted for secretion. The study was made using the two monokaryotic protoclones (mkPC9 and mkPC15) whose genomes had been previously sequenced and annotated in a collaborative project carried out with the Joint Genome Institute. These two protoclones contain the two nuclei present in the commercial dikaryotic strains dkN001. The results obtained showed that, surprisingly, both strains differ in their lignocellulose degrading genomic capabilities. mkPC9 have less CAZy genes annotated, especially in the Glycosil hydrolases (GH) class. Nevertheless, mkPC9 grows better than mkPC15 on lignocellulosic substrates and has a higher enzyme secretion capacity when growing in the presence of wood. The transcription of the genes coding for secretable proteins was studied by RNAseq analysis and we could conclude that, whereas the genome profile of the secretome was similar in the two strains, the corresponding transcriptome profiles were different between them and in different culture conditions and we observed a concentrated transcriptional activity in few genes per function and an increased importance of the glycosil hydrolases and proteins without a functional classification. These results highlight the importance of adding additional data to the gene lists produced by genome sequence analysis for gaining a more accurate picture of the biological process under study. P. ostreatus secretes a huge variety of lignocellulose degrading enzymes when cultured in the presence of wood. More than 20% of them lack a known enzymatic function. Transcriptome analysis noted the importance of these proteins, further confirmed by proteomics. Using domain structure prediction, we were able to give an insight about the possible role of several proteins, including a xylanase and a AA10 LPMO. This chapter is a version of the manuscript entitled Comparative and transcriptional analysis of the predicted secretome in the lignocellulose‐degrading basidiomycete fungus Pleurotus ostreatus published in Environmental Microbiology. Finally, in the fourth chapter, mass spectrometry analyses were used to confirm the presence of these enzymes acting on the lignocellulosic substrates. We compared the proteins secreted by the two monokaryons studied in the bioinformatics analysis with that of dikaryon that contains the two nuclei present in them. Interestingly, monokaryons behave in a very different manner; mkPC15 showed a weakest production of lignocellulose degrading enzymes than mkPC9 and dkN001 when cultured using wood as a carbon source. Moreover, dkN001 was able to secrete more plant cell wall decomposing enzymes, correlating with their superior capacity to grow on lignocellulosic substrates. Furthermore, the three strains were cultured in three different media using with glucose, wood or both (glucose and wood) as a carbon source. As expected, we identify a higher number of lignocellulose degrading enzymes in wood-containing media, especially glycosyl-hydrolases, carbohydrate esterases and polysaccharide lyases. Fungal lignocellulose degradation is the result of the synergistic action of several enzymes. These thesis improve our overall understanding of plant biomass degradation as a step to achieve the goal of using biomass as a sustainable source of energy to support future needs.

Alfaro Sánchez, Manuel

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