Biochemical and structural studies of recombinant basidiomycetes AA9 LPMOs

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform an oxidative cleavage of glycosidic bonds. The uncommon and remarkable LPMOs' active site is composed of a single copper atom coordinated by three nitrogen ligands, a structural motif known as histidine brace. The nitrogen ligands are provided by the amino group and side chain of an N-terminal His, and a third comes from another histidine side chain, giving rise to a T-shaped copper coordination geometry. The identification of LPMOs activity and its cooperativity with classical hydrolytic enzymes was a paradigmatic change in the enzymatic conversion of lignocellulosic polysaccharides, especially for the viability of biorefineries. LPMOs are currently known to be present in the genomes of viruses and many taxa across the Tree of Life, and they are classified within the families AA9, AA10, AA11, AA13, AA14, AA15, AA16 and AA17 in the CAZy (Carbohydrate-Active enZymes) database (cazy.org). Members of the family AA9 (LPMO9s) are exclusive and widely distributed among fungi with different lifestyles. In this regard, while some white-rot basidiomycetes exceed 30 genes encoding LPMO9s, ubiquitous ascomycetes such as Neurospora crassa and Aspergillus spp. harbor around 15 and 8 genes, respectively. The differential co-expression/secretion of LPMO9s along with other plant cell wall degrading enzymes has been verified in response to the substrate, especially lignocellulosic materials. Functional studies have revealed that LPMO9s are active not only on cellulose and cellooligosaccharides but also on a range of hemicellulosic substrates. Among them, the activity on tamarind xyloglucan is the most commonly verified. The number of AA9 LPMOs predicted in Basidiomycetes genomes is much higher than displayed by Ascomycetes, however, functional studies regarding substrate preference and regioselectivity are still limited. According to the CAZy database, three LPMOs from the white rots Heterobasidion irregulare, Lentinus similis and Phanerochaete chrysosporium, and two from the brown rot Gloeophyllum trabeum were characterized so far. Here, a proteomic approach will be used to investigate the enzymatic arsenal of CAZymes from Picnoporus coccineus and Trametes versicolor, focusing on the search for novel AA9 LPMOs. For this, the growth and protein secretion pattern of both white-rot fungi on different lignocellulosic substrates will be investigated. The results will provide a global understanding of the strategies employed by these microorganisms in the degradation of different lignocellulosic substrates and allow the raising of a series of target LPMOs for production using the A. oryzae engineered strain, along with biochemical and structural studies.