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Lignin is a highly abundant aromatic polymer in nature, but its controlled cleavage or cross-linking is a major challenge and currently hindering industrial applicability. Laccase (L) and laccase/mediator systems (LMS) are promising tools for enzymatic lignin modification, but to date, their overall reaction outcome is hard to predict and control. This research aimed to understand the reactivity of native and sulfonated β-O-4 linked lignin structures in L and LMS treatments. Trametes versicolor laccase, and the mediator hydroxybenzotriazole (HBT) were used, and reaction products were analyzed using UHPLC-MS n and MALDI-TOF-MS. Polymerization was observed for both the native and sulfonated phenolic compounds, suggesting that sulfonation does not affect radical coupling of the phenolic lignin subunits. In contrast, sulfonation of the non-phenolic lignin structure prevented C α oxidation and cleavage by L/HBT, which was explained by an increased C α -H bond dissociation energy of ∼10 kcal mol -1 upon sulfonation. Overall, our results indicate that lignin sulfonation drives the overall outcome of LMS incubations towards polymerization.

Lignin degradation by white-rot fungi is an essential step in terrestrial carbon cycling and has great potential for biotechnological applications. Selective white-rot fungi have been recognized for their ability to effectively delignify lignocellulose, but the underlying mechanisms, particularly in situ, have largely remained elusive to date. In this work, we elucidate specific degradation routes of β-O-4 aryl ethers in actual lignocellulosic biomass for the industrially relevant selective white-rot fungus Ceriporiopsis subvermispora. Multidimensional NMR and py-GC-MS analyses together with enzymatically synthesized model compounds enabled, for the first time, the identification of various diagnostic lignin cleavage products in residual wheat straw. Our results support that in situ ligninolysis by C. subvermispora is initiated by single-electron transfer, which then cascades into the cleavage of Cα-Cβ, Cβ-O, and O-4-aryl bonds of β-O-4 aryl ethers. The high abundance of 1-(benzyl)-2,3-dihydroxypropan-1-ones indicated that β-O-|4 cleavage is a more important pathway than previously considered. Our approach highlights key diagnostic substructures for providing mechanistic insight into fungal ligninolysis.

The white-rot fungus Ceriporiopsis subvermispora delignifies plant biomass extensively and selectively and, therefore, has great biotechnological potential. We previously demonstrated that after 7 weeks of fungal growth on wheat straw 70% w/w of lignin was removed and established the underlying degradation mechanisms via selectively extracted diagnostic substructures. In this work, we fractionated the residual (more intact) lignin and comprehensively characterized the obtained isolates to determine the susceptibility of wheat straw lignin's structural motifs to fungal degradation. Using 13C IS pyrolysis gas chromatography-mass spectrometry (py-GC-MS), heteronuclear single quantum coherence (HSQC) and 31P NMR spectroscopy, and size-exclusion chromatography (SEC) analyses, it was shown that β-O-4′ ethers and the more condensed phenylcoumarans and resinols were equally susceptible to fungal breakdown. Interestingly, for β-O-4′ ether substructures, marked cleavage preferences could be observed: β-O-4′-syringyl substructures were degraded more frequently than their β-O-4′-guaiacyl and β-O-4′-tricin analogues. Furthermore, diastereochemistry (threo > erythro) and γ-acylation (γ-OH > γ-acyl) influenced cleavage susceptibility. These results indicate that electron density of the 4′-O-coupled ring and local steric hindrance are important determinants of oxidative β-O-4′ ether degradation. Our findings provide novel insight into the delignification mechanisms of C. subvermispora and contribute to improving the valorization of lignocellulosic biomass.

BACKGROUND White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal-treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py-GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of −0.84 versus −0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents – among others, due to interference of fungal biomass. CONCLUSION Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Socie

Background: This study investigated the ruminal degradability of various wheat straw types by the white-rot fungi Ceriporiopsis subvermispora (CS) and Lentinula edodes (LE). Different cultivars (CV) of wheat straw at different maturity stages (MS) were treated with the fungi for 7weeks and assessed for chemical composition and in vitro gas production (IVGP). Results: Both fungi showed a more pronounced degradation of lignin on a more mature straw (MS3; 89.0%) in comparison with the straw harvested at an earlier stage (MS1; 70.7%). Quantitative pyrolysis coupled to gas chromatography and mass spectrometry, using 13C lignin as an internal standard 13C-IS Py-GC/MS revealed that lignin inmoremature straw was degraded and modified to a greater extent. In contrast, cellulose was less degraded in MS3, as compared to MS1 (8.3% versus 14.6%). There was no effect of different MS on the IVGP of the fungus-treated straws. Among the different straw cultivars, the extent of lignin degradation varied greatly (47% to 93.5%). This may explain the significant (P < 0.001) effect of cultivar on the IVGP of the fungal-treated straws. Regardless of the factors tested, both fungi were very capable of improving the IVGP of all straw types by 15.3% to 47.6%, (as compared to untreated straw), with CS performing better than LE – on different MS (33.6% versus 20.4%) and CVs (43.2% versus 29.1%). Conclusion: The extent of lignin degradation caused by fungal treatment was more pronounced on the more mature and lignified straw, while variable results were obtained with different cultivars. Both fungi were capable of improving the IVGP of various straw types.

Ammonia treatment of lignocellulose improves carbohydrate degradability, however, low ammonia dose treatment effects and mechanisms are hardly considered. This study describes low dose ammonia treatment of wheat straw in a statistical design of experiments (Taguchi design) to evaluate the effects of ammonia concentration, treatment time and the Solid:Liquid ratio on structure, composition and enzymatic degradability of the residual fractions. The results showed that low ammonia concentration (≤2 w/w % NH3) resulted in a high carbohydrate recovery (>80%) coupled enzymatic hydrolysis of 50% of xylan and 40% of glucan of the treated material using a (hemi-) cellulase enzyme cocktail. This effect coincidences with the relative decrease in ferulic acid by 10% and coumaric acid by more than 50% analysed via pyrolysis-GC–MS, measured as 4-vinyl-phenol and 4-vinyl-guaiacol, respectively. Our findings show that lowering ammonia concentration increased the effect of treatment time on the enzymatic degradability of the residual fraction.

BACKGROUND: White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal-treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py-GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS: Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of −0.84 versus −0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents – among others, due to interference of fungal biomass. CONCLUSION: Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

With the ever-advancing lignocellulose valorization strategies, lignin analyses need to advance as well. However, lignin quantification still heavily relies on unspecific, time- and sample-consuming gravimetric, and spectrophotometric analyses. Here, we demonstrate that lignin isolates from uniformly 13C-labeled wheat straw, willow, and douglas fir serve as "ideal" internal standards for pyrolysis gas chromatography high-resolution mass spectrometry (py-GC-HR-MS) analyses of plant biomass, allowing the accurate and precise quantification and structural characterization of lignin in grasses, hardwoods, and softwoods. The 13C lignin internal standards were comprehensively structurally characterized by HSQC NMR and py-GC-HR-MS analyses, and their application for lignin quantification was validated in biomass model systems and in actual plant biomass. For all botanical origins and species, the lignin content was determined within 5% relative deviation of the Klason benchmark. These results establish the capability of the developed analytical platform to selectively quantify and structurally characterize lignin simultaneously and demonstrate a valuable addition to the lignin analysis toolbox.

Agaricus bisporus consumes carbohydrates contained in wheat straw based compost used for commercial mushroom production. Double substituted arabinoxylan is part of the ~40% of the compost polysaccharides that are not degraded by A. bisporus during its growth and development. Genes encoding α-1,3-L-arabinofuranosidase (AXHd3) enzymes that act on xylosyl residues doubly substituted with arabinosyl residues are absent in this mushroom forming fungus. Here, the AXHd3 encoding hgh43 gene of Humicola insolens was expressed in A. bisporus with the aim to improve its substrate utilization and mushroom yield. Transformants secreted active AXHd3 in compost as shown by the degradation of double substituted arabinoxylan oligomers in an in vitro assay. However, carbohydrate composition and degree of arabinosyl substitution of arabinoxylans were not affected in compost possibly due to inaccessibility of the doubly substituted xylosyl residues., BT/Industrial Microbiology

Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor, alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.

In this study, two acid catalysts, acetic acid (HAc) and sulfuric acid (H2SO4), were compared in thermal pretreatments of corn stover, in particular to assess the less understood fate of lignin. HAc-insoluble lignin, analyzed by pyrolysis GC–MS, showed decreasing levels (%) of Cα-oxidized (from 3.7 ± 0.2 to 1.8 ± 0.1), propenyl (from 2.5 ± 0.1 to 1.0 ± 0.1), vinyl-G (from 34.5 ± 1.8 to 28.4 ± 0.9), vinyl-S (from 4.2 ± 0.2 to 3.7 ± 0.1) and methylated (from 4.9 ± 0.04 to 2.8 ± 0.1) lignin units at increasing HAc amounts. Concurrently, unsubstituted and vinyl-H units increased (from 7.5 ± 0.5 to 11.3 ± 0.2 and from 40.5 ± 1.9 to 49.9 ± 0.9, respectively). Similar trends were seen for residual lignin in H2SO4 catalyzed pretreatments, although the composition differed from that of residual HAc-lignin. In particular, H2SO4-lignin showed slightly lower values (%) for unsubstituted (9.9 ± 0.2) and vinyl-H (45.7 ± 4.1) units, while Cα-oxidized (3.4 ± 0.4), propenyl (1.9 ± 0.1), vinyl-G (28.5 ± 0.9), vinyl-S (4.4 ± 0.6) and methylated (4.6 ± 0.2) lignin units remained higher compared to HAc-catalysis at similar pH values. Xylan yields and corresponding enzymatic conversions of the solids were similar regardless the type of acid. Our findings show that HAc in pretreatments decreased lignin complexity, possibly due to cleavage reactions, although subsequent recondensation reactions increased solid lignin yields, more than H2SO4, while removal of xylan and enzymatic conversion of solids were equal.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 μm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

The white-rot fungi Ceriporiopsis subvermispora (Cs), Pleurotus eryngii (Pe), and Lentinula edodes (Le) have been shown to be high-potential species for selective delignification of plant biomass. This delignification improves polysaccharide degradability, which currently limits the efficient lignocellulose conversion into biochemicals, biofuels, and animal feed. Since selectivity and time efficiency of fungal delignification still need optimization, detailed understanding of the underlying mechanisms at molecular level is required. The recently developed methodologies for lignin quantification and characterization now allow for the in-depth mapping of fungal modification and degradation of lignin and, thereby, enable resolving underlying mechanisms. Results Wheat straw treated by two strains of Cs (Cs1 and Cs12), Pe (Pe3 and Pe6) and Le (Le8 and Le10) was characterized using semi-quantitative py-GC–MS during fungal growth (1, 3, and 7 weeks). The remaining lignin after 7 weeks was quantified and characterized using 13C lignin internal standard based py-GC–MS and whole cell wall HSQC NMR. Strains of the same species showed similar patterns of lignin removal and degradation. Cs and Le outperformed Pe in terms of extent and selectivity of delignification (Cs ≥ Le >> Pe). The highest lignin removal [66% (w/w); Cs1] was obtained after 7 weeks, without extensive carbohydrate degradation (factor 3 increased carbohydrate-to-lignin ratio). Furthermore, though after treatment with Cs and Le comparable amounts of lignin remained, the structure of the residual lignin vastly differed. For example, Cα-oxidized substructures accumulated in Cs treated lignin up to 24% of the total aromatic lignin, a factor two higher than in Le-treated lignin. Contrarily, ferulic acid substructures were preferentially targeted by Le (and Pe). Interestingly, Pe-spent lignin was specifically depleted of tricin (40% reduction). The overall subunit composition (H:G:S) was not affected by fungal treatm

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a β-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.

Agaricus bisporus consumes carbohydrates contained in wheat straw based compost used for commercial mushroom production. Double substituted arabinoxylan is part of the ~40% of the compost polysaccharides that are not degraded by A. bisporus during its growth and development. Genes encoding α-1,3-L-arabinofuranosidase (AXHd3) enzymes that act on xylosyl residues doubly substituted with arabinosyl residues are absent in this mushroom forming fungus. Here, the AXHd3 encoding hgh43 gene of Humicola insolens was expressed in A. bisporus with the aim to improve its substrate utilization and mushroom yield. Transformants secreted active AXHd3 in compost as shown by the degradation of double substituted arabinoxylan oligomers in an in vitro assay. However, carbohydrate composition and degree of arabinosyl substitution of arabinoxylans were not affected in compost possibly due to inaccessibility of the doubly substituted xylosyl residues.

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave glycosidic bonds in polysaccharides. The ability of these copper enzymes to boost the degradation of lignocellulose has greatly stimulated research efforts and biocatalytic applications within the biorefinery field. Initially found as oxidizing recalcitrant substrates, such as chitin and cellulose, it is now clear that LPMOs cleave a broad range of oligo- and poly-saccharides and make use of various electron-donating systems. Herein, substrate specificities and electron-donating systems of fungal LPMOs are summarized. A closer look at LPMOs as part of the fungal enzyme machinery might provide insights into their role in fungal growth and plant-pathogen interactions to further stimulate the search for novel LPMO applications.

Background The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. Results Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. Conclusions The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

Background: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. Results: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. Conclusions: The G. thermodenitrificans T12 endo-xylana

Background: Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. Results: Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. Conclusions: The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.