Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression.

BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far. RESULTS: Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes. CONCLUSION: The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides.

Continuous photometric activity assays for lytic polysaccharide monooxygenase-Critical assessment and practical considerations.

Lytic polysaccharide monooxygenase (LPMO) is a monocopper-dependent enzyme that cleaves glycosidic bonds by using an oxidative mechanism. In nature, they act in concert with cellobiohydrolases to facilitate the efficient degradation of lignocellulosic biomass. After more than a decade of LPMO research, it has become evident that LPMOs are abundant in all domains of life and fulfill a diverse range of biological functions. Independent of their biological function and the preferred polysaccharide substrate, studying and characterizing LPMOs is tedious and so far mostly relied on the discontinuous analysis of the solubilized reaction products by HPLC/MS-based methods. In the absence of appropriate substrates, LPMOs can engage in two off-pathway reactions, i.e., an oxidase and a peroxidase-like activity. These futile reactions have been exploited to set up easy-to-use continuous spectroscopic assays. As the natural substrates of newly discovered LPMOs are often unknown, widely applicable, simple, reliable, and robust spectroscopic assays are required to monitor LPMO expression and to perform initial biochemical characterizations, e.g., thermal stability measurements. Here we provide detailed descriptions and practical protocols to perform continuous photometric assays using either 2,6-dimethoxyphenol (2,6-DMP) or hydrocoerulignone as colorimetric substrates as a broadly applicable assay for a range of LPMOs. In addition, a turbidimetric measurement is described as the currently only method available to continuously monitor LPMOs acting on amorphous cellulose.