The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

Light-driven lytic polysaccharide monooxygenase catalysis mediated by Type I photosensitizers.

The use of light as abundant, renewable, and clean energy source to boost lytic polysaccharide monooxygenase (LPMO) reactions represents an exciting and yet under-explored opportunity. Herein we demonstrated that photosensitizers, commonly used in photodynamic therapy, which act through the photocatalytic Type I mechanism can drive the oxidation of PASC by LPMOs, whereas Type II photosensitizers are not capable of promoting the LPMO activity. We analyzed Type I and Type II photosensitizers (methylene blue and tetraiodide salt of meso-tetrakis-(4-N-methylpyridyl) porphyrin, respectively) and demonstrated that, even without an addition of external reductant, Type I was capable of boosting Thermothelomyces thermophila MtLPMO9A activity in the presence of light. We also evaluated the photobiosystem in the presence and/or absence of molecular oxygen (O2) and hydrogen peroxide (H2O2), and investigated the role of superoxide radical in the methylene blue fueled reactions. Furthermore, we demonstrated that sodium bisulfite (NaHSO3), a chemical scavenger of H2O2, acts by safeguarding the enzyme from oxidative damage caused by accumulation of H2O2 early in photosensitizer-driven LPMO reactions. Finally, the results of the present work demonstrated that light-driven LPMO reactions mediated photodynamic therapy (PDT) Type I photosensitizers, which also includes molecules such as curcumin and riboflavin, is a general phenomenon.

Photodegradation in terrestrial ecosystems.

The first step in carbon (C) turnover, where senesced plant biomass is converted through various pathways into compounds that are released to the atmosphere or incorporated into the soil, is termed litter decomposition. This review is focused on recent advances of how solar radiation can affect this important process in terrestrial ecosystems. We explore the photochemical degradation of plant litter and its consequences for biotic decomposition and C cycling. The ubiquitous presence of lignin in plant tissues poses an important challenge for enzymatic litter decomposition due to its biological recalcitrance, creating a substantial bottleneck for decomposer organisms. The recognition that lignin is also photolabile and can be rapidly altered by natural doses of sunlight to increase access to cell wall carbohydrates and even bolster the activity of cell wall degrading enzymes highlights a novel role for lignin in modulating rates of litter decomposition. Lignin represents a key functional connector between photochemistry and biochemistry with important consequences for our understanding of how sunlight exposure may affect litter decomposition in a wide range of terrestrial ecosystems. A mechanistic understanding of how sunlight controls litter decomposition and C turnover can help inform management and other decisions related to mitigating human impact on the planet.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

H2 O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid.

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H2 O2 at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H2 O2 to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H2 O2 (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.

Molecular mechanism of the chitinolytic peroxygenase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei.

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation.

Identification and functional study of AA11 family polysaccharide monooxygenase genes in filamentous fungus Podospora anserina.

The lytic polysaccharide monooxygenase (LPMO) in the auxiliary active protein family (AA family) catalyzes the oxidative depolymerization of various refractory carbohydrates including cellulose, chitin and starch. While accumulating studies investigate the enzymology of LPMO, the research on the inactivation of LPMO genes has been rarely explored. In this study, five LPMO genes PaLPMO11A (Pa_4_4790), PaLPMO11B (Pa_1_5310), PaLPMO11C (Pa_2_7840), PaLPMO11D (Pa_2_8610) and PaLPMO11E (Pa_3_9420) of the AA11 family in the filamentous fungus Podospora anserina were knocked out by homologous recombination. Single mutants ΔPaLPMO11A (ΔA), ΔPaLPMO11B (ΔB), ΔPaLPMO11C (ΔC), ΔPaLPMO11D (ΔD) and ΔPaLPMO11E (ΔE) were constructed, and then all polygenic mutants were constructed via genetic crosses. The differences in the growth rate and sexual reproduction between wild type and mutant strains were observed on different carbon source media. The alteration of oxidative stress and cellulose degradation ability were found on DAB and NBT staining and cellulase activity determination. These results implicated that LPMO11 genes play a key role in the growth, development, and lignocellulose degradation of P. anserina. The results showed that the spore germination efficiency, growth rate and reproductive capacity of mutant strains including ΔBΔCΔE, ΔAΔBΔCΔE, ΔAΔCΔDΔE and ΔAΔBΔCΔDΔE was significantly decreased on different cellulose carbon sources and the remaining strains have no difference. The reduced utilization of various carbon sources, the growth rate, the spore germination rate, the number of fruiting bodies, the normal fruiting bodies, the shortened life span and the ability to degrade cellulose were found in strains which all five genes in the PaLPMO11 family were deleted. However, the strain still had 45% cellulase activity compared to wild type. These results suggest that LPMO11 genes may be involved in the growth and development, sexual reproduction, senescence and cellulose degradation of P. anserina. This study provides information for systematically elucidating the regulatory mechanism of lignocellulose degradation in filamentous fungus P. anserina.

Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs.

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, ß-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

Chitin-Active Lytic Polysaccharide Monooxygenases Are Rare in Cellulomonas Species.

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the ß-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on ß-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here, we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six T. terrestris AA9 (TtAA9) LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. TtLPMO9A and TtLPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, TtLPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. TtLPMO9A, TtLPMO9E, TtLPMO9G, and TtLPMO9T could degrade tamarind xyloglucan and, with the exception of TtLPMO9T, beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs, as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates. IMPORTANCE The discovery of LPMOs in 2010 has revolutionized the industrial biotechnology field, mainly by increasing the efficiency of cellulolytic enzyme cocktails. Nonetheless, the biological purpose of the multiplicity of LPMO-encoding genes in filamentous fungi has remained an open question. Here, we address this point by showing that six AA9 LPMOs from a single fungal strain have various substrate preferences and activities on tested cellulosic and hemicellulosic substrates, including several native xylan substrates. Importantly, several of these activities could only be detected when using copolymeric substrates that likely resemble plant cell walls more than single fractionated polysaccharides do. Our results suggest that LPMOs have evolved to contribute to the degradation of different complex structures in plant cell walls where different biomass polymers are closely associated. This knowledge together with the elucidated novel xylanolytic activities could aid in further optimization of enzymatic cocktails for efficient degradation of lignocellulosic substrates and more.

Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis.

BACKGROUND: Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context. RESULTS: In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs. CONCLUSION: The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

Structural perturbations of substrate binding and oxidation state changes in a lytic polysaccharide monooxygenase.

LPMOs are enzymes which catalyse the oxidation of a C-H bond within polysaccharides, leading to their oxidative cleavage. To achieve this, LPMOs employ highly reactive oxidising intermediates, the generation of which is likely coupled to substrate binding to the enzyme. The nature of this coupling is unknown. Here we report a statistical comparison for four three-dimensional structures of an AA9 LPMO crystallised in the same space group but in different oxidation and substrate-binding states, to determine which significant structural perturbations occur at the enzyme upon either oxidation state change or the binding of substrate. In a novel step, we determine the global random error associated with the positional coordinates of atoms using the method of moments to ascertain the statistical estimators of Gaussian distributions of pairwise RMS differences between individual atoms in different structures. The results show that a change in the oxidation state of the copper leads to no significant structural changes, and that binding of the substrate leads to a single change in the conformation of a tryptophan residue. This tryptophan has previously been identified as part of a charge transfer pathway between the active site and the external surface of the protein, and the structural change identified herein may be part of the substrate-enzyme coupling mechanism.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Oxidative cleavage of cellulose in the horse gut.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. RESULTS: We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro. CONCLUSIONS: The oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

Genomic and Proteomic Study of Andreprevotia ripae Isolated from an Anthill Reveals an Extensive Repertoire of Chitinolytic Enzymes.

Chitin is an abundant natural polysaccharide that is hard to degrade because of its crystalline nature and because it is embedded in robust co-polymeric materials containing other polysaccharides, proteins, and minerals. Thus, it is of interest to study the enzymatic machineries of specialized microbes found in chitin-rich environments. We describe a genomic and proteomic analysis of Andreprevotia ripae, a chitinolytic Gram-negative bacterium isolated from an anthill. The genome of A. ripae encodes four secreted family GH19 chitinases of which two were detected and upregulated during growth on chitin. In addition, the genome encodes as many as 25 secreted GH18 chitinases, of which 17 were detected and 12 were upregulated during growth on chitin. Finally, the single lytic polysaccharide monooxygenase (LPMO) was strongly upregulated during growth on chitin. Whereas 66% of the 29 secreted chitinases contained two carbohydrate-binding modules (CBMs), this fraction was 93% (13 out of 14) for the upregulated chitinases, suggesting an important role for these CBMs. Next to an unprecedented multiplicity of upregulated chitinases, this study reveals several chitin-induced proteins that contain chitin-binding CBMs but lack a known catalytic function. These proteins are interesting targets for discovery of enzymes used by nature to convert chitin-rich biomass. The MS proteomic data have been deposited in the PRIDE database with accession number PXD025087.

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin.

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and ß-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants-JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18-indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.