Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates

Abstract

Background: Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood. Results:In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from Podospora anserina (PaLPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of PaLPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of PaLPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network. Conclusions:Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and nonoxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity

“…The obtained K M -and k cat -values of NcLPMO9C and the variant LPMOs were similar to previously published values [36]. In contrast to the recently published work by Chalak and coworkers [32], we were able to successfully produce an active LPMO variant with a truncated linker and CBM1 right after the catalytic domain. However, this modification resulted in a partial reduction of the thermostability.…”

“…When looking at the regioselectivity on cellulosic substrates, NcLPMOs 9F, 9C and 9M share the same product pattern as previously published [19]. In contrast to Chalak and coworkers [32] no change in the regioselectivity of the enzyme was observed when removing the CBM1. However, recent efforts to elucidate the role of a family 2 CBM linked to LPMO10C from Streptomyces coelicolor revealed no change in regioselectivity upon truncation of the CBM2 from the enzyme [26].…”

“…In addition to a catalytic module, around one fifth of fungal LPMOs feature a C-terminal CBM1 connected via a flexible linker of variable length and amino acid composition [2,31]. CBM1 is an important module empowering cellulose binding of many hydrolytic cellulose-degrading enzymes [32].…”

“…A similar binding role of this CBM1 for LPMOs was demonstrated by isothermal calorimetry (ITC), which measured a forty times lower dissociation constant (K d ) for the complex between the CBM1 of NcLPMO9C and regenerated amorphous cellulose (RAC) than for the complex between the catalytic domain of NcLPMO9C and RAC [24]. Interestingly, removal of the CBM1 from the C1/C4 oxidizing Podospora anserina LPMO9H, not only negatively affected binding and activity, but also resulted in a shift in regioselectivity towards predominantly C1 oxidation [32]. Similarly, other studies reported that truncation of the linker and CBM1 of LPMOs affect both the substrate binding and the formed products [24,26].…”

Current understanding of substrate specificity and regioselectivity of LPMOs

“…Domain similarity network analysis has shown the correlation between the additional domains and the substrate specificity of the full enzymes (Book et al 2014;Zhou et al 2019b). CBM truncation studies have been reported for both LPMO9s and LPMO10s (Chalak et al 2019;Courtade et al 2018;Crouch et al 2016;Forsberg et al 2016;Laurent et al 2019). Comparison of the performance of LPMOs with and without CBMs have shown that, deletion of CBMs reduced LPMO's binding capacity to crystalline substrates, especially at low substrate concentrations.…”