Florian Csarman

Lignocellulolytic enzyme cocktails for biomass saccharification are commonly produced using solid-state fermentation (SSF) or submerged fermentation (SmF), whereas sequential solid state and submerged (SSF→SmF) co-cultivation remain poorly explored for synergistic fungal enzyme production. In this study, enzyme production strategies (SSF, SmF, and SSF→SmF) were evaluated using Fusarium oxysporum f. sp. lagenariae JCM 9293, Laetiporussulphureus DSM 1014, and Irpexlacteus DSM 9595 cultivated on dilute acid-pretreated common reed and ryegrass in shaking flasks. Sequential co-cultivation, where I. lacteus was grown during SSF followed by F. oxysporum during the SmF phase, yielded the highest specific enzyme activities in shake flasks after 9 days, reaching 5.24 U mg⁻¹ cellulase, 26.1 U mg⁻¹ CMC-ase, and 43.1 U mg⁻¹ xylanase. Enzyme production on common reed exceeded ryegrass by 1.2–2.5 fold, consistent with its higher glucan content (52 %). Partial diafiltration further enriched xylanase activity to 61.8 U mg⁻¹. Scale-up experiments in a stirred-tank bioreactor and an air-lift bioreactor confirmed a successful SSF→SmF transfer. The highest productivity was achieved in a horizontal rotating tubular bioreactor, which achieved up to 75 % of shake-flask enzyme productivity. Lower aeration and shear conditions in the stirred-tank and air-lift bioreactors reduced performance. These results demonstrate that time separated and optimized fungal co-cultivation enables the scalable production of lignocellulolytic enzyme cocktails from abundant wetland biomass, thus supporting sustainable biorefinery applications.

The genome of Fusarium solani, a well‐known plant pathogen, encodes various lytic polysaccharide monooxygenases (LPMOs) involved in plant biomass degradation in combination with cellobiose dehydrogenase (CDH). To investigate the auxiliary role of the recently expressed and characterized class III CDH from F. solani (FsCDH), this enzyme was tested in combination with the well‐characterized AA9C from Neurospora crassa (NcAA9C). Steady‐state and stopped‐flow methods as well as electrochemical measurements demonstrate how FsCDH efficiently transfers electrons to NcAA9C, with a rapid, observed heme reoxidation rate constant of 129 s⁻¹. In comparison to ascomycete class II CDHs, the H₂O₂ production by FsCDH is insufficient to promote LPMO activity. However, a cyclic cascade between NcAA9C and FsCDH was found. NcAA9C reaction products showed a high catalytic efficiency as FsCDH substrates, with K_M values close to its natural substrate cellobiose. This reaction was further investigated by a real time measurement, where FsCDH and NcAA9C were incubated with phosphoric acid‐swollen cellulose and the reaction was sustained over a long period without the addition of an external reductant. The new class III CDH is similar to other CDH classes, except its very low reactivity with molecular oxygen, pointing towards a different function in Ascomycota than class II CDH. These findings contribute to the better understanding of oxidative cellulose degradation by fungi and thus, to potential biotechnological applications for the sustainable use of biomass.

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.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD‐containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure‐based engineering. The electron transfer kinetics of wild‐type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped‐flow spectrophotometry and structural effects were studied by small‐angle X‐ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway – its mutation reducing the interdomain electron transfer 10‐fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron‐transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X‐ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair‐distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS‐based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H₂O₂ rather than O₂ is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H₂O₂-based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H₂O₂ concentration in their vicinity with a piezo-controlled H₂O₂ microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H₂O₂. The results point towards a high catalytic efficiency of LPMO at a low H₂O₂ concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.