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In the opinion paper by Zhao et al. ‘Making the biochemical conversion of lignocellulose more robust’, the authors claim that ‘…lignocellulose biorefinery is conceptually wrong’. In response, we argue that this claim itself has already been proved wrong by several companies., In the opinion paper by Zhao et al. ‘Making the biochemical conversion of lignocellulose more robust’, the authors claim that ‘…lignocellulose biorefinery is conceptually wrong’. In response, we argue that this claim itself has already been proved wrong by several companies.

The present invention relates to an enzyme exhibiting histidine N-methyltransferase activity, a microbial cell comprising a gene encoding such enzyme, and use of this enzyme for producing a target peptide or protein having a methylated N-terminal histidine residue.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the N epsilon 2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported. In this study, a diverse library of 100 plant extracts was screened for LPMO activity-modulating effects. By employing protein crystallography and LC-MS we successfully identified a natural LPMO inhibitor. Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites, one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1. The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts., Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported. In this study, a diverse library of 100 plant extracts was screened for LPMO activity-modulating effects. By employing protein crystallography and LC–MS, we successfully identified a natural LPMO inhibitor. Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC–MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1. The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin–protein interactions.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharide substrates oxidatively. First discovered because of their action on recalcitrant crystalline substrates (chitin and cellulose), a number of LPMOs are now reported to act on soluble substrates, including oligosaccharides. However, crystallographic complexes with oligosaccharides have been reported for only a single LPMO so far, an enzyme from the basidiomycete fungus Lentinus similis (LsAA9_A). Here we present a more detailed comparative study of LsAA9_A and an LPMO from the ascomycete fungus Collariella virescens (CvAA9_A) with which it shares 41.5% sequence identity. LsAA9_A is considerably more thermostable than CvAA9_A, and the structural basis for the difference has been investigated. We have compared the patterns of oligosaccharide cleavage and the patterns of binding in several new crystal structures explaining the basis for the product preferences of the two enzymes. Obtaining structural information about complexes of LPMOs with carbohydrates has proven to be very difficult in general judging from the structures reported in the literature thus far, and this can be attributed only partly to the low affinity for small substrates. We have thus evaluated the use of differential scanning fluorimetry as a guide to obtaining complex structures. Furthermore, an analysis of crystal packing of LPMOs and glycoside hydrolases corroborates the hypothesis that active site occlusion is a very significant problem for LPMO-substrate interaction analysis by crystallography, due to their relatively flat and extended substrate binding sites.

Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. PACE is a sensitive and simple tool for studying polysaccharide structure or quantity and also has applications in the investigation of enzyme specificity.

Background: The bioconversion of lignocellulosic feedstocks to ethanol is being commercialised, but further process development is required to improve their economic feasibility. Efficient saccharification of lignocellulose to fermentable sugars requires oxidative cleavage of glycosidic linkages by lytic polysaccharide monooxygenases (LPMOs). However, a proper understanding of the catalytic mechanism of this enzyme class and the interaction with other redox processes associated with the saccharification of lignocellulose is still lacking. The in-use stability of LPMO-containing enzyme cocktails is increased by the addition of catalase implying that hydrogen peroxide (H 2O 2) is generated in the slurry during incubation. Therefore, we sought to characterize the effects of enzymatic and abiotic sources of H 2O 2 on lignocellulose hydrolysis to identify parameters that could improve this process. Moreover, we studied the abiotic redox reactions of steam-pretreated wheat straw as a function of temperature and dry-matter (DM) content. Results: Abiotic reactions in pretreated wheat straw consume oxygen, release carbon dioxide (CO 2) to the slurry, and decrease the pH. The magnitude of these reactions increased with temperature and with DM content. The presence of LPMO during saccharification reduced the amount of CO 2 liberated, while the effect on pH was insignificant. Catalase led to increased decarboxylation through an unknown mechanism. Both in situ-generated and added H 2O 2 caused a decrease in pH. Conclusions: Abiotic redox processes similar to those that occur in natural water-logged environments also affect the saccharification of pretreated lignocellulose. Heating of the lignocellulosic material and adjustment of pH trigger rapid oxygen consumption and acidification of the slurry. In industrial settings, it will be of utmost importance to control these processes. LPMOs interact with the surrounding redox compounds and redirect abiotic electron f

Detailed studies of enzymatic hydrolysis of the abundant but recalcitrant biopolymers cellulose and chitin has led to a revised understanding of enzymatic decomposition of polysaccharides. Copper-enzymes now classified as lytic polysaccharide monooxygenases (LPMOs) were first annotated as either glycosyl hydrolases or as binding domains without catalytic activity. It was shown that enzymes at the time classified as family GH61 are copper-dependent enzymes that cleaves glucosidic bonds when provided with plant derived cofactors. The first crystal structures of Cu-LPMO, the observation of and evidence for a plausible role of N-methylation of the copper coordinating N-terminal histidine in fungal LPMOs, the first study of binding of dioxygen to reduced Cu-LPMO, the first atomic structures of a starch active LPMO and the first atomic structures of a LPMO-oligosaccharide complex are contributions that have led to significant advances in the scientific understanding. In addition, use of LPMOs for industrial saccharification of lignocellulose was studied in detail and the importance of controlling the oxidative process was emphasised. A positive effect of adding catalase was attributed to prolonged enzyme half-life and thus the first evidence of oxidative deactivation of cellulolytic enzymes in this context. Furthermore, it was discovered that catalytic activity of LPMOs can be improved many fold when photo excited pigments, rather than any previously tested reducing agents, was used as the electron donating cofactor for the enzyme. The discovery of LPMOs and the understanding that they constitute the “first wave of attack” by microbial organisms on the most recalcitrant natural polysaccharides, highlights the need to reflect on the specific roles of LPMOs in biology. Many good and relevant research questions are awaiting the scientific community. Based on current knowledge, the presence of LPMOs should be anticipated in all microbial systems where the degradation

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.

Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks.

Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. Results: Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. Conclusions: This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage method prior to grass pr

The pH within cell wall pores of filter paper fibers and hemp fibers was assessed by Fluorescence Ratio Imaging (FRIM). It was found that the Donnan effect affected the pH measured within the fibers. When the conductivity of the added liquid was low (0. 7 mS), pH values were lower within the cell wall than in the bulk solution. This was not the case at high conductivity (22 mS). The occurrence of the Donnan effect allowed the pH values within pores in normal regions of the cell wall to be compared to the pH in regions with misaligned microfibrils (dislocations) when FRIM was carried out in a low conductivity solution. Surprisingly, no pH difference was observed between normal regions and dislocations, suggesting that pore sizes within the two different regions are approximately the same. In another experiment the Donnan effect was shown to have an effect on hydrolysis of hydrothermally pretreated wheat straw only when conducted in a low conductivity solution and only for xylanase, not cellulases. The hydrolysis experiments indicate that under typical conditions where conductivity is high, the Donnan effect does not lower the pH close to the substrate to an extent that affects enzymatic activity during hydrolysis of lignocellulose.

Grass biomass is a prospective type of lignocellulosic biomass for bioenergy and fuel production, but the low dry matter in grass at harvest calls for new pretreatment strategies for cellulosic conversion. In this study, ensiling was tested as a biological pretreatment method of the high yielding grass variety Festulolium Hykor. The biomass was harvested in four cuts over a growing season. Three important factors of ensiling: biomass composition, dry matter (DM) at ensiling, and inoculation of lactic acid bacteria, were assessed in relation to subsequent enzymatic cellulose hydrolysis. The organic acid profile after ensiling was dependant on the composition of the grass and the DM, rather than on the inocula. High levels of organic acids, notably lactic acid, produced during ensiling improved enzymatic cellulose convertibility in the grass biomass. Ensiling of less mature grass gave higher convertibility. Low DM at ensiling (<25%) resulted in the highest cellulose convertibilities, which ranged from 32 to 70% of the available cellulose in the four cuts after ensiling. The study confirms that ensiling can enhance cellulose convertibility of green biomass, and provides new insight to ensiling as a biological pretreatment method for green biomass conversion.

Most secondary plant cell walls contain irregular regions known as dislocations or slip planes. Under industrial biorefining conditions dislocations have recently been shown to play a key role during the initial phase of the enzymatic hydrolysis of cellulose in plant cell walls. In this review we chart previous publications that have discussed the structure of dislocations and their susceptibility to hydrolysis. The supramolecular structure of cellulose in dislocations is still unknown. However, it has been shown that cellulose microfibrils continue through dislocations, i.e. dislocations are not regions where free cellulose ends are more abundant than in the bulk cell wall. In more severe cases cracks between fibrils form at dislocations and it is possible that the increased accessibility that these cracks give is the reason why hydrolysis of cellulose starts at these locations. If acid or enzymatic hydrolysis of plant cell walls is carried out simultaneously with the application of shear stress, plant cells such as fibers or tracheids break at their dislocations. At present it is not known whether specific carbohydrate binding modules (CBMs) and/or cellulases preferentially access cellulose at dislocations. From the few studies published so far it seems that no special type of CBM is involved. In one case an endoglucanase was found to preferably bind to dislocations

Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of the reducing ends of sugars with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. PACE is a sensitive and simple tool for studying polysaccharide structure or quantity and also has applications in the investigation of enzyme specificity.