Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section.

Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers. The method can reveal potential differences in fiber properties along the cross-sectional profile of natural or man-made cellulose fibers. In this analytical approach, carbonyl groups are labeled with a carbonyl selective fluorescence label (CCOA), after which thin fiber layers are sequentially dissolved with the solvent system DMAc/LiCl (9% w/v) and analyzed with size exclusion chromatography coupled with light scattering and fluorescence detection. The analysis of these fractions allowed for the recording of the changes in the chemical structure across the layers, resulting in a detailed cross-sectional profile of the different functionalities and molecular weight distributions. The method was optimized and tested in practice with LPMO (lytic polysaccharide monooxygenase)-treated cotton fibers, where it revealed the depth of fiber modification by the enzyme.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities.

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Oxidation of cellulose fibers using LPMOs with varying allomorphic substrate preferences, oxidative regioselectivities, and domain structures.

Lytic polysaccharide monooxygenases (LPMOs) are excellent candidates for enzymatic functionalization of natural polysaccharides, such as cellulose or chitin, and are gaining relevance in the search for renewable biomaterials. Here, we assessed the cellulose fiber modification potential and catalytic performance of eleven cellulose-active fungal AA9-type LPMOs, including C1-, C4-, and C1/C4-oxidizing LPMOs with and without CBM1 carbohydrate-binding modules, on cellulosic substrates with different degrees of crystallinity and polymer chain arrangement, namely, Cellulose I, Cellulose II, and amorphous cellulose. The potential of LPMOs for cellulose fiber modification varied among the LPMOs and depended primarily on operational stability and substrate binding, and, to some extent, also on regioselectivity and domain structure. While all tested LPMOs were active on natural Cellulose I-type fibers, activity on the Cellulose II allomorph was almost exclusively detected for LPMOs containing a CBM1 and LPMOs with activity on soluble hemicelluloses and cello-oligosaccharides, for example NcAA9C from Neurospora crassa. The single-domain variant of NcAA9C oxidized the cellulose fibers to a higher extent than its CBM-containing natural variant and released less soluble products, indicating a more dispersed oxidation pattern without a CBM. Our findings reveal great functional variation among cellulose-active LPMOs, laying the groundwork for further LPMO-based cellulose engineering.

A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres.

Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications. An obstacle for wide implementation of LPMOs in tailored cellulose chemistry is the lack of suitable techniques to precisely monitor the LPMO impact on the polymer. Soluble oxidized cello-oligomers can be quantified using chromatographic and mass-spectrometric techniques. A considerable portion of the oxidized sites, however, remain on the insoluble cellulose fibres, and their quantification is difficult. Here, we describe a method for the simultaneous quantification of oxidized sites on cellulose fibres and changes in their molar mass distribution after treatment with LPMOs. The method is based on quantitative, heterogeneous, carbonyl-selective labelling with a fluorescent label (CCOA) followed by cellulose dissolution and size-exclusion chromatography (SEC). Application of the method to reactions of seven different LPMOs with pure cellulose fibres revealed pronounced functional differences between the enzymes, showing that this CCOA/SEC/MALS method is a promising tool to better understand the catalytic action of LPMOs.

Functional characterization of a lytic polysaccharide monooxygenase from Schizophyllum commune that degrades non-crystalline substrates.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that use O2 or H2O2 to oxidatively cleave glycosidic bonds. LPMOs are prevalent in nature, and the functional variation among these enzymes is a topic of great interest. We present the functional characterization of one of the 22 putative AA9-type LPMOs from the fungus Schizophyllum commune, ScLPMO9A. The enzyme, expressed in Escherichia coli, showed C4-oxidative cleavage of amorphous cellulose and soluble cello-oligosaccharides. Activity on xyloglucan, mixed-linkage ß-glucan, and glucomannan was also observed, and product profiles differed compared to the well-studied C4-oxidizing NcLPMO9C from Neurospora crassa. While NcLPMO9C is also active on more crystalline forms of cellulose, ScLPMO9A is not. Differences between the two enzymes were also revealed by nuclear magnetic resonance (NMR) titration studies showing that, in contrast to NcLPMO9C, ScLPMO9A has higher affinity for linear substrates compared to branched substrates. Studies of H2O2-fueled degradation of amorphous cellulose showed that ScLPMO9A catalyzes a fast and specific peroxygenase reaction that is at least two orders of magnitude faster than the apparent monooxygenase reaction. Together, these results show that ScLPMO9A is an efficient LPMO with a broad substrate range, which, rather than acting on cellulose, has evolved to act on amorphous and soluble glucans.

Revisiting the AA14 family of lytic polysaccharide monooxygenases and their catalytic activity.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose-xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re-evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose-associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.

Hemicellulolytic enzymes in lignocellulose processing.

Lignocellulosic biomass is the most abundant source of carbon-based material on a global basis, serving as a raw material for cellulosic fibers, hemicellulosic polymers, platform sugars, and lignin resins or monomers. In nature, the various components of lignocellulose (primarily cellulose, hemicellulose, and lignin) are decomposed by saprophytic fungi and bacteria utilizing specialized enzymes. Enzymes are specific catalysts and can, in many cases, be produced on-site at lignocellulose biorefineries. In addition to reducing the use of often less environmentally friendly chemical processes, the application of such enzymes in lignocellulose processing to obtain a range of specialty products can maximize the use of the feedstock and valorize many of the traditionally underutilized components of lignocellulose, while increasing the economic viability of the biorefinery. While cellulose has a rich history of use in the pulp and paper industries, the hemicellulosic fraction of lignocellulose remains relatively underutilized in modern biorefineries, among other reasons due to the heterogeneous chemical structure of hemicellulose polysaccharides, the composition of which varies significantly according to the feedstock and the choice of pretreatment method and extraction solvent. This paper reviews the potential of hemicellulose in lignocellulose processing with focus on what can be achieved using enzymatic means. In particular, we discuss the various enzyme activities required for complete depolymerization of the primary hemicellulose types found in plant cell walls and for the upgrading of hemicellulosic polymers, oligosaccharides, and pentose sugars derived from hemicellulose depolymerization into a broad spectrum of value-added products.

Engineering cellulases for conversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs). As enzymes are a considerable cost factor, there is great interest in finding or engineering improved and robust cellulases, with higher activity and stability, easy expression, and minimal product inhibition. This review addresses relevant engineering targets for cellulases, discusses a few notable cellulase engineering studies of the past decades and provides an overview of recent work in the field.

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.

Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression.

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a "green enzyme," laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda's gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7-8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.

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.

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.

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa.

Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi, where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose and some also act on hemicelluloses, primarily other (substituted) ß-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. crassa are active on both cellulose and cellulose-associated glucuronoxylan but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that NcLPMO9F preferentially cleaves xylan when acting on a cellulose-beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, from Malbranchea cinnamomea, showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. IMPORTANCE Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain unclear. Here, we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose-glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.

In situ measurements of oxidation-reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose.

BACKGROUND: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. RESULTS: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation-reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. CONCLUSION: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation-reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.

Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi.

The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth's carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes. In this study, we present comparable quantitative protein abundance values across five species and five diverse substrates. The results allow for direct comparison of fungal adaptation to the different substrates, give indications as to the substrate specificity of individual carbohydrate-active enzymes (CAZymes), and reveal proteins of unknown function that are co-expressed with CAZymes. Based on the results, we present a quantitative comparison of 34 lytic polysaccharide monooxygenases (LPMOs), which are crucial enzymes in biomass deconstruction.

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives.

Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

Heterologous expression of lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) are relatively new enzymes that have been discovered 10 years ago. LPMOs comprise a diverse group of enzymes which play a pivotal role in the depolymerization of sugar-based biopolymers including cellulose, hemicellulose, chitin, and starch. Their mechanism of action relies on the correct coordination of a copper ion in the active site, which is partly composed of the N-terminal histidine. Therefore, correct secretion and folding of these copper-enzymes is fundamental for obtaining a catalytic activity. LPMOs occur in all kingdoms of life; they have been found in viruses, bacteria and eukaryotes, including fungi, plants and animals. In many cases, using homologous expression of these proteins is not feasible and an alternative organism, which can be cultured and is able to heterologously express the protein of interest, is required for studying enzyme properties. Therefore, we made an extensive compilation of expression techniques used for LPMOs the expression and characterization of which have been reported to date. In the current review, we provide a summary of the different techniques, including expression hosts and vectors, secretion methods, and culturing conditions, that have been used for the overexpression and production of this important class of enzymes at laboratory scale. Herein, we compare these techniques and assess their advantages and disadvantages.

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the ß-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications.

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost-effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large-scale protein production, and extensive host-specific post-translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum-based transient expression technology, and this recombinant enzyme (TrCel7Arec ) was compared with the native fungal enzyme (TrCel7Anat ) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N-terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O-mannosylation in the plant host as compared with the native protein. In general, the purified full-length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate-binding properties, which can be attributed to larger N-glycans and lack of O-glycans in TrCel7Arec . All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization.

The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers' physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.