Biomass-degrading Enzymes Research Articles

Mimicking the enzymatic plant cell wall hydrolysis mechanism for the degradation of polyethylene terephthalate

Plastic pollution presents a global challenge, impacting ecosystems, wildlife, and economies. Polyethylene terephthalate (PET), widely used in products like bottles, significantly contributes to this issue due to poor waste collection. In recent years, there has been increasing interest in plant biomass-degrading enzymes for plastic breakdown, due to the structural and physicochemical similarities between natural and synthetic polymers. Filamentous fungi involved in hemicellulose degradation have developed a complex mode of action that includes not only enzymes but also biosurfactants; surface-active molecules that facilitate enzyme-substrate interactions. For this reason, this study aimed to mimic the mechanism of biomass degradation by repurposing plant cell wall degrading enzymes including a cutinase and three esterases to cooperatively contribute to PET degradation. Surfactants of different charge were also introduced in the reactions, as their role is similar to biosurfactants, altering the surface tension of the polymers and thus improving enzymes’ accessibility. Notably, Fusarium oxysporum cutinase combined with anionic surfactant exhibited a 2.3- and 1.6-fold higher efficacy in hydrolyzing amorphous and semi-crystalline PET, respectively. When cutinase was combined with either of two ferulic acid esterases, it resulted in complete conversion of PET intermediate products to TPA, increasing the overall product release up to 1.9– fold in presence of surfactant. The combination of cutinase with a glucuronoyl esterase demonstrated significant potential in plastic depolymerization, increasing degradation yields in semi-crystalline PET by up to 1.4-fold. The approach of incorporating enzyme cocktails and surfactants emerge as an efficient solution for PET degradation in mild reaction conditions, with potential applications in eco-friendly plastic waste management.

Co-production of sugars and aroma compounds from tobacco waste using biomass-degrading enzymes produced by Aspergillus brunneoviolaceus Ab-10.

Biomass-degrading enzymes produced by microorganisms have a great potential in the processing of agricultural wastes. In order to produce suitable biomass-degrading enzymes for releasing sugars and aroma compounds from tobacco scraps, the feasibility of directly using the scraps as a carbon source for enzyme production was investigated in this study. By comparative studies of ten fungal strains isolated from tobacco leaves, Aspergillus brunneoviolaceus Ab-10 was found to produce an efficient enzyme mixture for the saccharification of tobacco scraps. Proteomic analysis identified a set of plant biomass-degrading enzymes in the enzyme mixture, including amylases, hemicellulases, cellulases and pectinases. At a substrate concentration of 100g/L and enzyme dosage of 4mg/g, glucose of 17.6g/L was produced from tobacco scraps using the crude enzyme produced by A. brunneoviolaceus Ab-10. In addition, the contents of 23 volatile molecules, including the aroma compounds 4-ketoisophorone and benzyl alcohol, were significantly increased after the enzymatic treatment. The results provide a strategy for valorization of tobacco waste by integrating the production of biomass-degrading enzymes into the tobacco scrap processing system.

Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymatic activities, and transcriptomics on cellulose. From plate cultures, the Δxyr1 mutant had reduced growth on D-xylose, xylan, and cellulose, and from shake-flask cultures with cellulose, the Δxyr1 mutant had ~90% lower β-glucosidase activity, and no detectable β-xylosidase or cellulase activity. The comparison of the transcriptomes from 18 h shake-flask cultures on D-fructose, without a carbon source, and cellulose, showed major effects of XYR1 deletion whereby the Δxyr1 mutant on cellulose was transcriptionally most similar to the cultures without a carbon source. The cellulose induced 43 plant biomass-degrading CAZymes including xylanases as well as cellulases, and most of these had massively lower expression in the Δxyr1 mutant. The expression of a subset of carbon catabolic enzymes, other transcription factors, and sugar transporters was also lower in the Δxyr1 mutant on cellulose. In summary, T. harzianum XYR1 is the master regulator of cellulases and xylanases, as well as regulating carbon catabolic enzymes.

The Conformational Change of the L3 Loop Affects the Structural Changes in the Substrate Binding Pocket Entrance of β-Glucosidase

β-glucosidase (Bgl) hydrolyzes cellobiose to glucose, thereby releasing non-reducing terminal glucosyl residues. Bgl is an essential enzyme belonging to the biomass-degrading enzyme family, which plays a vital role in enzymatic saccharification during biofuel production. The four loops above the Bgl substrate-binding pocket undergo a conformational change upon substrate recognition. However, the structural dynamism of this loop and how it is conserved among Bgl family members remain unknown. Herein, to better understand the four loops above the substrate-binding pocket of Bgl, four Bgl crystal structures in Thermoanaerobacterium saccharolyticum (TsaBgl) were determined at 1.5–2.1 Å. The L1, L2, and L4 loops of TsaBgl showed a rigid conformation stabilized by their neighboring residues via hydrogen bonds and hydrophobic interactions. The TsaBgl L3 loop showed relatively high flexibility and two different N-terminal region conformations. The conformational change in the TsaBgl L3 loop induced a change in charge and shaped at the substrate-binding pocket entrance. The amino acid sequences and structures of the TsaBgl L1–4 loops were compared with other 45 Bgl proteins, and a diversity of the L2 and L3 loops was observed. Differences in amino acids and lengths of Bgls L2–L3 loop induced differences in the conformation and structure of the Bgls substrate-binding pocket entrance. These findings expand our knowledge on the molecular function of the loops in the Bgl enzyme family.

Transcriptomic profiling of Beauveria bassiana SAN01, an endophytic fungal entomopathogen, for the production of lignocellulosic enzymes

Beauveria bassiana, widely known for its ecological roles as an entomopathogen and as an endophyte, has recently been observed with a remarkable ability to utilize lignocellulosic biomass with the help of its active glycosyl hydrolases and other accessory enzymes. Hence, the capability of the fungus to produce different lignocellulosic biomass degrading enzymes was demonstrated quantitatively in this study. Furthermore, the transcriptional responses of B. bassiana during lignocellulose degradation in comparison with its well-studied endophytic condition were elucidated by comparative genome-wide transcriptomics using the NextSeq 550 system. The B. bassiana strain was recorded to produce enzymes including amylases, cellulases, glucanases, pectinases as well as various lignin and xylan degrading enzymes at significant levels ranging from 126.12 ± 7.71 U/L (laccase) to 295.42 ± 12.65 U/mL (xylanase). Subsequently, a clear molecular distinction was shown under the different tropic states as 458, 521 and 497 genes were found to be differently expressed in the fungus between the fermentation vs endophytic, fermentation vs control and endophytic vs control groups, respectively. The Carbohydrate Active Enzyme (CAZy) annotation of the transcriptome under fermentation conditions also showed the upregulation of many carbohydrases that were determined quantitatively earlier in the study. This study thus highlights the adaptive plasticity of B. bassiana for the deconstruction of plant biomass with the aim of enhancing its alternative industrial applications, especially in the production of biomass-degrading enzymes for biofuel production.

Comparative seasonal analysis of Eri silkworm (Samia ricini Donovan) gut composition: implications for lignocellulose degradation.

Conversion of biomass such as lignocelluloses to an alternative energy source can contribute to sustainable development. Recently, biomass-degrading enzymes are reported to be common resources in insect-microbe interacting systems. Northeast India harbors ample sericigenous insect resources which are exploited for their silk products. Samia ricini Donovan is an economically important poly-phytophagous silkmoth capable of digesting foliage from different plant species, suggesting the versatility of a robust gut system. Here, a gut bacterial profile was determined by 16S rRNA gene characterization across the holometabolous life cycle during the summer and winter seasons, revealing 3 phyla, 13 families, and 22 genera. Comparative analysis among the seasonal gut isolates revealed a high diversity in summer, predominated by the genus Bacillus due to its high occurrence in all developmental stages. Shannon's diversity index demonstrated the second and fourth instars of summer as well as the fifth instar of winter to be relatively better developmental stages for gut bacteria assembly. Bacterial community shifts in concert to host developmental changes were found to be apparent between early instars and late instars in summer, which differed from those of winter. Forty-three and twenty-nine gut bacterial isolates were found to be cellulolytic and xylanolytic enzyme producers, respectively. The present results illustrate the gut microbiota of S. ricini over the seasons and support the holometabolous life cycle effect as the most likely factor shaping the gut bacterial microbiota. These findings may provide leads for the development of new cleaner and environmentally friendly lignocellulose-degrading enzymes.

A High-Quality Genome Sequence of the Penicillium oxalicum 5-18 Strain Isolated from a Poplar Plantation Provides Insights into Its Lignocellulose Degradation.

Studies on the degradation of plant cell wall polysaccharides by fungal extracellular enzymes have attracted recent attention from researchers. Xylan, abundant in hemicellulose, that play great role in connection between cellulose and lignin, has seen interest in its hydrolytic enzymatic complex. In this study, dozens of fungus species spanning genera were isolated from rotting leaves based on their ability to decompose xylan. Among these isolates, a strain with strong xylanase-producing ability was selected for further investigation by genome sequencing. Based on phylogenetic analysis of ITS (rDNA internal transcribed spacer) and LSU (Large subunit 28S rDNA) regions, the isolate was identified as Penicillium oxalicum. Morphological analysis also supported this finding. Xylanase activity of this isolated P. oxalicum 5-18 strain was recorded to be 30.83 U/mL using the 3,5-dinitro-salicylic acid (DNS) method. Further genome sequencing reveals that sequenced reads were assembled into a 30.78 Mb genome containing 10,074 predicted protein-encoding genes. In total, 439 carbohydrate-active enzymes (CAZymes) encoding genes were predicted, many of which were associated with cellulose, hemicellulose, pectin, chitin and starch degradation. Further analysis and comparison showed that the isolate P. oxalicum 5-18 contains a diverse set of CAZyme genes involved in degradation of plant cell wall components, particularly cellulose and hemicellulose. These findings provide us with valuable genetic information about the plant biomass-degrading enzyme system of P. oxalicum, facilitating a further exploration of the repertoire of industrially relevant lignocellulolytic enzymes of P. oxalicum 5-18.

High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi.

Anaerobic fungi produce biomass-degrading enzymes and natural products that are important to harness for several biotechnology applications. Although progress has been made in the development of methods for extracting nucleic acids for genomic and transcriptomic sequencing of these fungi, most studies are limited in that they do not sample multiple fungal growth phases in batch culture. In this study, we establish a method to harvest RNA from fungal monocultures and fungal-methanogen co-cultures, and also determine an optimal time frame for high-quality RNA extraction from anaerobic fungi. Based on RNA quality and quantity targets, the optimal time frame in which to harvest anaerobic fungal monocultures and fungal-methanogen co-cultures for RNA extraction was 2-5 days of growth post-inoculation. When grown on cellulose, the fungal strain Anaeromyces robustus cocultivated with the methanogen Methanobacterium bryantii upregulated genes encoding fungal carbohydrate-active enzymes and other cellulosome components relative to fungal monocultures during this time frame, but expression patterns changed at 24-hour intervals throughout the fungal growth phase. These results demonstrate the importance of establishing methods to extract high-quality RNA from anaerobic fungi at multiple time points during batch cultivation.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source

BackgroundThe filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei.ResultsWe chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain.ConclusionsIn general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the β-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

BackgroundFilamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.ResultsA novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.ConclusionsThis PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Biomass-Degrading Enzyme(s) Production and Biomass Degradation by a Novel Streptomyces thermocarboxydus.

Modern society has a great challenge to decrease waste and minimize the adverse effects of wastes on the economy, environment, and individual health. Thus, this study focuses on the use of eight agro-wastes (banana peel, barley straw, canola straw, pomegranate peel, orange peel, pumpkin pulp+seeds, maple leaf, and brewer's spent grains) by a novel bacterium (Streptomyces thermocarboxydus) for enzymes production. Further, the study explored the subsequent degradation of those wastes by the bacterium. This bacterium was isolated from forest soil and identified as Streptomyces thermocarboxydus by 16S rRNA sequence analysis. The biodegrading capability of S. thermocarboxydus was determined by observing the clear zone around the colony cultured on the agar plate containing the different biomasses as sole carbon sources and calculating the substrate degradation ratios. Furthermore, scanning electron microscopy images of eight agro-wastes before and after bacterial treatment and weight loss of agro-wastes revealed the bacterium degraded the biomasses. The different trends of enzyme activities were observed for various wastes, and the maximum activity depended on the type of agro-wastes. Overall, S. thermocarboxydus was found to be a potential candidate for pectinase and xylanase production. The enzyme production varies with the concentration of the biomasses.

Electrochemical characterization of a family AA10 LPMO and the impact of residues shaping the copper site on reactivity

Research on enzymes for lignocellulose biomass degradation has progressively increased in recent years due to the interest in taking advantage of this natural resource. Among these enzymes are the lytic polysaccharide monooxygenases (LPMOs) that oxidatively depolymerize crystalline cellulose using a reactive oxygen species generated in a reduced mono‑copper active site. The copper site comprises of a highly conserved histidine-brace, providing three equatorial nitrogen ligands, whereas less conserved residues close to the copper contribute to shaping and confining the site. The catalytic copper site is exposed to the solvent and to the crystalline substrates, and as so, the influence of the copper environment on LPMO properties, including the redox potential, is of great interest. In the current work, a direct electrochemical study of an LPMO (ScLPMO10C) was conducted allowing to retrieve kinetic and thermodynamic data associated with the redox transition in the catalytic centre. Moreover, two residues that do not bind to the copper but shape the copper sites were mutated, and the properties of the mutants were compared with those of the wild-type enzyme. The direct electrochemical studies, using cyclic voltammetry, yielded redox potentials in the +200 mV range, well in line with LPMO redox potentials determined by other methods. Interestingly, while the mutations hardly affected the formal redox potential of the enzyme, they drastically affected the reactivity of the copper site and enzyme functionality.

Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates.

Understanding the biodiversity and genetics of gut microbiomes has important implications for host physiology and industrial enzymes, whereas most studies have been focused on bacteria and archaea, and to a lesser extent on fungi and viruses. One group, still underexplored and elusive, is ciliated protozoa, despite its importance in shaping microbiota populations. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies from 11 abundant morphogenera. With these genomes, we resolved the taxonomic and phylogenetic framework that revised the 22 morphospecies into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Comparative genomic analyses revealed that extensive horizontal gene transfers and gene family expansion provided rumen ciliate species with a broad array of carbohydrate-active enzymes (CAZymes) to degrade all major kinds of plant and microbial carbohydrates. In particular, the genomes of Diplodiniinae and Ophryoscolecinae species encode as many CAZymes as gut fungi, and ~80% of their degradative CAZymes act on plant cell-wall. The activities of horizontally transferred cellulase and xylanase of ciliates were experimentally verified and were 2-9 folds higher than those of the inferred corresponding bacterial donors. Additionally, the new ciliate dataset greatly facilitated rumen metagenomic analyses by allowing ~12% of the metagenomic sequencing reads to be classified as ciliate sequences.

Characterization of a novel recombinant halophilic β-glucosidase of Trichoderma harzianum derived from Hainan mangrove

Backgroundβ-glucosidase is an important biomass-degrading enzyme and plays a vital role in generating renewable biofuels through enzymatic saccharification. In this study, we analyzed the transcriptome of Trichoderma harzianum HTASA derived from Hainan mangrove and identified a new gene encoding β-glucosidase Bgl3HB. And the biochemically characterization of β-glucosidase activity was performed.ResultsBgl3HB showed substantial catalytic activity in the pH range of 3.0–5.0 and at temperatures of 40 ℃-60 ℃. The enzyme was found quite stable at 50 ℃ with a loss of only 33.4% relative activity after 240 min of heat exposure. In addition, all tested metal ions were found to promote the enzyme activity. The β-glucosidase activity of Bgl3HB was enhanced by 2.12-fold of its original activity in the presence of 5 M NaCl. Surprisingly, Bgl3HB also showed a remarkable ability to hydrolyze laminarin compared to other measured substrates. Enzyme efficiency was examined in the sugarcane bagasse saccharification processes, in which Bgl3HB with 5 M NaCl worked better supplementing Celluclast 1.5L than the commercial Novozyme 188 ascertained it as an admirably suited biocatalyst for the utilization of agricultural waste. In this work, this is the first report of a halophilic β-glucosidase from Trichoderma harzianum, and represents the β-glucosidase with the highest known NaCl activation concentration. And adding 5 M NaCl could enhance saccharification performance even better than commercial cellulase.ConclusionsThese results show that Bgl3HB has great promise as a highly stable and highly efficient cellulase with important future applications in the industrial production of biofuels.

Exploring the multi-level regulation of lignocellulases in the filamentous fungus Trichoderma guizhouense NJAU4742 from an omics perspective

BackgroundFilamentous fungi are highly efficient at deconstructing plant biomass by secreting a variety of enzymes, but the complex enzymatic regulation underlying this process is not conserved and remains unclear.ResultsIn this study, cellulases and xylanases could specifically respond to Avicel- and xylan-induction, respectively, in lignocellulose-degrading strain Trichoderma guizhouense NJAU4742, however, the differentially regulated cellulases and xylanases were both under the absolute control of the same TgXyr1-mediated pathway. Further analysis showed that Avicel could specifically induce cellulase expression, which supported the existence of an unknown specific regulator of cellulases in strain NJAU4742. The xylanase secretion is very complex, GH10 endoxylanases could only be induced by Avicel, while, other major xylanases were significantly induced by both Avicel and xylan. For GH10 xylanases, an unknown specific regulator was also deduced to exist. Meanwhile, the post-transcriptional inhibition was subsequently suggested to stop the Avicel-induced xylanases secretion, which explained the specifically high xylanase activities when induced by xylan in strain NJAU4742. Additionally, an economical strategy used by strain NJAU4742 was proposed to sense the environmental lignocellulose under the carbon starvation condition, that only slightly activating 4 lignocellulose-degrading genes before largely secreting all 33 TgXyr1-controlled lignocellulases if confirming the existence of lignocellulose components.ConclusionsThis study, aiming to explore the unknown mechanisms of plant biomass-degrading enzymes regulation through the combined omics analysis, will open directions for in-depth understanding the complex carbon utilization in filamentous fungi.

Disruption of vacuolar protein sorting receptor gene Poxvps10 improves cellulolytic enzyme production by Penicillium oxalicum

Penicillium oxalicum can secrete numerous of plant biomass-degrading enzymes, but limited information is available regarding the mechanisms associated with their secretion. In the Golgi-to-vacuole pathway, the type I transmembrane receptor Vps10p is involved in the sorting of the soluble vacuolar proteins and can also target recombinant and aberrant proteins from the Golgi to the vacuole for degradation. Here, we used the combination of phenotypic characterization and comparative secretome analysis to explore the effect of disruption of the vps10 gene in P. oxalicum (Poxvps10) on endogenous cellulolytic enzyme secretion. The study found that PoxVps10p is required for the targeting and delivery of vacuolar PoxCpyA to the vacuole in P. oxalicum. Poxvps10p deletion enhances extracellular protein and cellulase production by P. oxalicum when the cells are grown on a cellulosic substrate (wheat bran and Avicel). Furthermore, secretome analysis revealed higher relative amount of cellulases, lytic polysaccharide monooxygenase and post-translational modification-related proteins in the ΔPoxvps10 mutant than in the wild-type (WT) strain, which may explain the higher cellulase production by the ΔPoxvps10 than the WT strain. This study thus provides a new target for manipulating the secretory pathway to enhance the cellulolytic enzyme production.

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus.

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Genome sequence and Carbohydrate Active Enzymes (CAZymes) repertoire of the thermophilic Caldicoprobacter algeriensis TH7C1T

BackgroundOmics approaches are widely applied in the field of biology for the discovery of potential CAZymes including whole genome sequencing. The aim of this study was to identify protein encoding genes including CAZymes in order to understand glycans-degrading machinery in the thermophilic Caldicoprobacter algeriensis TH7C1T strain.ResultsCaldicoprobacter algeriensis TH7C1T is a thermophilic anaerobic bacterium belonging to the Firmicutes phylum, which grows between the temperatures of 55 °C and 75 °C. Next generation sequencing using Illumina technology was performed on the C. algeriensis strain resulting in 45 contigs with an average GC content of 44.9% and a total length of 2,535,023 bp. Genome annotation reveals 2425 protein-coding genes with 97 ORFs coding CAZymes. Many glycoside hydrolases, carbohydrate esterases and glycosyltransferases genes were found linked to genes encoding oligosaccharide transporters and transcriptional regulators; suggesting that CAZyme encoding genes are organized in clusters involved in polysaccharides degradation and transport. In depth analysis of CAZomes content in C. algeriensis genome unveiled 33 CAZyme gene clusters uncovering new enzyme combinations targeting specific substrates.ConclusionsThis study is the first targeting CAZymes repertoire of C. algeriensis, it provides insight to the high potential of identified enzymes for plant biomass degradation and their biotechnological applications.

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases.

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.