Discovery and applications of family AA9 lytic polysaccharide monooxygenases

Auxililary activity family 9 lytic polysaccharide monooxygenases (abbreviated as AA9s or LPMO9s) are fungal mono-copper enzymes capable of oxidatively cleaving various plant cell wall oligo- and/or polysaccharides. LPMO9s are key components of lignocellulolytic enzyme cocktails used in today’s biorefineries to break down biomass into fermentable sugars. Highly stable enzymes with novel functions are of great interest to improve enzymatic biorefinery processes and their economic feasibility. Genome sequencing of an industrially relevant fungus, Thermothielavioides terrestris LPH172, revealed 411 putative carbohydrate-active enzyme (CAZy) domains. Transcriptomic analysis indicated that the fungus upregulated numerous LPMO9 genes in concert with canonical cellulase and hemicellulase encoding genes to degrade lignocellulose. Nuanced co-upregulation was detected for LPMO9 genes and those encoding other redox-active CAZymes. Six strongly upregulated TtLPMO9 genes were heterologously expressed and functionally characterized using cellulosic and hemicellulosic substrates. These studies showed that the multitude of LPMO9 genes provided the fungus with different functions, including previously unknown cleavage of cellulose-associated spruce arabinoglucuronoxylan and acetylated birch glucuronoxylan. In a related study, xylanolytic LPMO9 activity was revealed or enhanced by debranching xylans enzymatically, which likely assumed a rigid and stretched xylan conformation that associated with cellulose to increase accessibility to LPMO9s. LPMOs have unique oxidative powers which render them advantageous for various biorefinery applications. A C1-oxidizing TtLPMO9G was found to increase the amount of carboxyl groups on sulfated cellulose nanocrystals by 10%, without any extensive degradation of the crystals. The functional groups thus generated were used for proof-of-concept crosslinking, which could aid in the production of bio-based materials. In another application, addition of TaLPMO9A to a benchmark LPMO-poor cellulolytic cocktail was shown to improve saccharification yields of mildly pretreated spruce substrates. The final glucose and xylose yields were increased by up to 1.6- and 1.5-fold, respectively, illustrating how LPMO9s can be exploited in the saccharification of these notoriously recalcitrant substrates

Carboxylation of sulfated cellulose nanocrystals by family AA9 lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) from the auxiliary activity 9 (AA9) family act on cellulose through an oxidative mechanism that improves cellulose saccharification in concert with other cellulolytic enzymes. Degradation and solubilization of cellulose chains are known to take place when various cellulose hierarchies, fibers, nanofibers, and cellulose nanocrystals (CNCs) are subjected to LPMOs, either alone or in combination with other cellulose acting enzymes. The use of LPMOs to modify and prepare CNCs has been proposed mostly in top-down synthesis from larger hierarchies. Here, we attempted a direct surface modification of CNCs with LPMOs with the aim of investigating the role played by the charged sulfate groups on CNCs. Sulfate half-ester\ua0groups are introduced during the preparation of CNCs from cellulose using sulfuric acid. It has been proposed that the charged sulfate groups hinder the binding of enzymes or affinity of charged reactants on the surface and hence reduce enzymatic and chemical reaction efficiency. We demonstrate the modification of commercial sulfated CNCs using a family AA9 LPMO. Conductometric titration and spectrometric characterization of the oxidized particles indicate that carboxylation of up to 10% was possible without degradation of the crystals. Unexpectedly, the carboxyl groups could only be introduced to the crystals containing\ua0sulfate groups, while desulfated crystals remained unfunctionalized. This was deemed to be due to that the sulfate groups limit the adsorption of the enzymes and hence modulate the cuts facilitated by the enzymes on the surface. This limits the release of chains from the surface and enables the carboxylation of the insoluble substrate rather than the release of the solubilized chains. This study highlights the importance of analyzing both the solid and soluble reaction products to gain insights into the oxidation mechanism. We demonstrated that 10% functionalization suffices for the use of CNCs in coupling chemistry

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases

Background: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions. Results: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with\ua0Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose–xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose–xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO–xylan interaction, because debranching changes the architecture of the xylan surface. Conclusions: Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials

Genomic and transcriptomic analysis of the thermophilic lignocellulose-degrading fungus Thielavia terrestris LPH172

Background: Biomass-degrading enzymes with improved activity and stability can increase substrate saccharification and make biorefineries economically feasible. Filamentous fungi are a rich source of carbohydrate-active enzymes (CAZymes) for biomass degradation. The newly isolated LPH172 strain of the thermophilic Ascomycete Thielavia terrestris has been shown to possess high xylanase and cellulase activities and tolerate low pH and high temperatures. Here, we aimed to illuminate the lignocellulose-degrading machinery and novel carbohydrate-active enzymes in LPH172 in detail. Results: We sequenced and analyzed the 36.6-Mb genome and transcriptome of LPH172 during growth on glucose, cellulose, rice straw, and beechwood xylan. 10,128 predicted genes were found in total, which included 411 CAZy domains. Compared to other fungi, auxiliary activity (AA) domains were particularly enriched. A higher GC content was found in coding sequences compared to the overall genome, as well as a high GC3 content, which is hypothesized to contribute to thermophilicity. Primarily auxiliary activity (AA) family 9 lytic polysaccharide monooxygenase (LPMO) and glycoside hydrolase (GH) family 7 glucanase encoding genes were upregulated when LPH172 was cultivated on cellulosic substrates. Conventional hemicellulose encoding genes (GH10, GH11 and various CEs), as well as AA9 LPMOs, were upregulated when LPH172 was cultivated on xylan. The observed co-expression and co-upregulation of genes encoding AA9 LPMOs, other AA CAZymes, and (hemi)cellulases point to a complex and nuanced degradation strategy. Conclusions: Our analysis of the genome and transcriptome of T. terrestris LPH172 elucidates the enzyme arsenal that the fungus uses to degrade lignocellulosic substrates. The study provides the basis for future characterization of potential new enzymes for industrial biomass saccharification

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

Novel AA9 LPMOs from the thermophilic fungus Thielavia terrestris LPH 172

For making biomass saccharification more efficient, industry is lookinginto thermophilic microorganisms for production of enzymes withimproved thermostability. One such organism is the fungus Thielaviaterrestris with alfalfa straw hydrolysis optima around 40 and 60\ub0C (Berkaet al., 2011). We isolated the ascomycete T. terrestris strain LPH 172 fromcompost in Vietnam and fully sequenced its genome. We have alsocharacterized the strain as acidophilic (growing at pH 1) and thermophilic(50\ub0C). High cellulase and xylanase activities at pH 3 and 70\ub0C were alsoobserved. Full genome sequencing revealed 10 128 genes,of which according to dbCAN2 380 encode for putative CAZymes and ofthose 18 for AA9 LPMOs. We also obtained transcriptomics data fromgrowth on five different substrates: glucose, cellulose (Avicel), beechwoodxylan, corncob xylan and rice straw. From differential and absolute geneexpression data analysis, we have selected nine putative AA9 LPMOenzymes that showed upregulation on growth on either Avicel or rice strawcompared to growth at glucose. For further characterization of these nineAA9s, they will be cloned and produced in Pichia pastoris and analyzed forprocessing simple and complex biomasses