Production of C1-oxidized cello-oligosaccharides by a fungal Lytic Polysaccharide Monooxygenase

GARRIDO M; LANDONI M; GHIO S; ONTAÑON ORNELLA M.; COUTO A; WIRTH S; CAMPOS E

Congreso; 3rd Argentinian Symposium on Glycobiology; 2019

Lignocellulosic biomass has high potential as a renewable resource for the production of fuels and chemicals.However, its natural resistance to microbial and enzymatic deconstruction makes its breakdown a slow and expensive process.A new class of secreted enzymes, known as lytic polysaccharide monooxygenases (LPMOs) was recently identified due to its boosting effect on enzymatic polysaccharide conversion by oxidizing glucose carbon atoms contained in cellulose, disrupting its crystalline structure and making it more accessible for glycosyl hydrolases. Fungal LPMOs belong to family AA9 and can be classified based on their regioselectivity (preferred site of oxidation), as: type 1, which only oxidize C1; type2, which only oxidize C4; and type 3, which can oxidize both C1 and C4. In this work, the coding sequence (960 bp) of an LPMO from the white rot fungi Pycnoporus sanguienus, named AA9Ps3, was cloned with its own signal sequence in fusion with a C-terminal 6-His tag in the expression vector pPIC9 and expressed in theyeast Pichia pastoris. The recombinant enzyme was purified from the culture broth supernatant. Activity of AA9Ps3 on Phosphoric Acid Swollen Cellulose (PASC) resulted in the productionof oxidized cello-oligosaccharides, with degrees of polymerization 2 to 5, detected by HPAEC-PAD and MALDI-TOF MS. The m/z relation of the products obtained indicate the formation of aldonic acids, which correspond to the oxidation of glucose C1 atoms. AA9Ps3 can be added to cellulolytic cocktails to improve cellulose bioconversion