Ninety percent of lignocellulose-degrading fungi contain genes encoding lytic polysaccharide monooxygenases (LPMOs). These enzymes catalyze the initial oxidative cleavage of recalcitrant polysaccharides after activation by an electron donor. Understanding the source of electrons is fundamental to fungal physiology and will also help with the exploitation of LPMOs for biomass processing. Using genome data and biochemical methods, we characterized and compared different extracellular electron sources for LPMOs: cellobiose dehydrogenase, phenols procured from plant biomass or produced by fungi, and glucose-methanol-choline oxidoreductases that regenerate LPMO-reducing diphenols. Our data demonstrate that all three of these electron transfer systems are functional and that their relative importance during cellulose degradation depends on fungal lifestyle. The availability of extracellular electron donors is required to activate fungal oxidative attack on polysaccharides.

BackgroundRecent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification.ResultsFour pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH.ConclusionsP. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

Background: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that cleave polysaccharides. Results: We describe a novel LPMO and use a range of analytical methods to characterize its activity. Conclusion: Cellulose and cello-oligosaccharides are cleaved by oxidizing the sugar at the nonreducing end in the C4 position. Significance: This study provides unequivocal evidence for C4 oxidation of the nonreducing end sugar and demonstrates a novel LPMO substrate specificity.

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

Background: Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.Results: Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.Conclusion: Critical features of surface morphology for LPMO synergy with cellulases are recognized.Significance: Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.