PSAA9A, a C1-specific AA9 lytic polysaccharide monooxygenase from the white rot Basidiomycete Pycnoporus sanguineus

LANDONI, MALENA; COUTO, ALICIA; CAMPOS, ELEONORA; SABBADIN, FEDERICO; BRUCE, NEIL; GARRIDO, MERCEDES; VALACCO, MARÍA PIA; WIRTH, SONIA

Buenos Aires

Congreso; Reunión Conjunta SAIB-SAMIGE; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. Among LPMOs, those belonging to family AA9 have been described to act exclusively on cellulose, oxidizing glucose carbon atoms in positions C1, C4 or both. In this work, we studied two AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus: PsAA9A and PsAA9B. Both were successfully produced as recombinant secreted proteins in Pichia pastoris. The correct processing of the pre-proteins and the presence of the conserved His1 from LPMOs in the mature enzymes were verified by mass spectrometry. PsAA9A was active on cellulosic substrates, with a C1-specific oxidizing mechanism generating native and oxidized cellooligosaccharides in the presence of external electron donors. It was active in a pH range from 5 to 7.5 and a temperature range from 30 to 60°C. As expected, PsAA9A was not active on several non-cellulosic substrates (such as xylan, glucomannan and α-chitin) but we did observe peaks compatible with oxidized chitooligosaccharides (CHOS) with DP 5 to 10 released from squid β-chitin, an activity not yet reported for LPMOs of family AA9. Conversely, we did not observe activity for PsAA9B, under any of the conditions tested. PsAA9A showed synergistic activity with a commercial GH1 β-glucosidase, a GH5 endoglucanase, and a GH6 cellobiohydrolase II, a result in accordance with previous transcriptomic and secretomic data from biomass-grown fungus, where PsAA9A was co-expressed with several GHs, including proteins from families GH5 and GH6. We did not observe an increase of GH7 cellobiohydrolase I (CBH I) activity on PASC by addition of PsAA9A when small phenolic electron donor, such as gallic acid, was present. Moreover, GH7 activity was lower in the presence of gallic acid (without PsAA9A). These results were in accordance with previous reports that described the inhibition of GH activity by phenolic compounds, which was higher for CBH I than for endoglucanases or β-glucosidases. This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus.