Lignocellulose-degrading Enzymes Research Articles

Use of lignocellulose-degrading enzyme bio-cemented soils in the construction industry

In their activity, termites break down biomass into glucose, forming cementitious compounds. This results in rainfall-resistant mounds for their shelters, which interests the construction industry. This study explored the potential application of the abundant naturally bio-cemented termite mound soils in highway construction. Experimental investigations compared these mound soils to surrounding soils, revealing that mound soils are 68.2% finer in particle size distribution and have 81.4% lower organic content. Atterberg limits indicated 14.6% higher plasticity and a 5.3% increase in specific gravity for mound soils. Atomic absorption spectroscopy showed no significant difference in chemical composition. Proctor tests indicated that the compaction characteristics of the soils were comparable, confirming their engineering viability without modifications. Mound soils exhibited a twofold increase in unconfined compressive strength and a 33.9% increment in California Bearing Ratio strength. One-way analysis of variance tests confirmed these differences with p-value ranges of 0.0056–0.0361 below the 0.05 significance threshold. The study advocates utilising eco-friendly naturally bio-cemented mound soils in highway construction, meeting minimum standards. Mound soils from one acre can construct up to 2,500 m highway, optimising resource use, promoting sustainability by repurposing natural materials, and significantly reducing carbon dioxide emissions. Further research on soil improvement using lignocellulose-degrading enzymes is recommended.

Novel Feruloyl Esterase for the Degradation of Polyethylene Terephthalate (PET) Screened from the Gut Microbiome of Plastic-Degrading Mealworms (Tenebrio Molitor Larvae).

Mealworms (Tenebrio molitor) larvae can degrade both plastics and lignocellulose through synergistic biological activities of their gut microbiota because they share similarities in chemical and physical properties. Here, a total of 428 genes encoding lignocellulose-degrading enzymes were screened from the gut microbiome of T. molitor larvae to identify poly(ethylene terephthalate) (PET)-degrading activities. Five genes were successfully expressed in E. coli, among which a feruloyl esterase-like enzyme named TmFae-PETase demonstrated the highest PET degradation activity, converting PET into MHET (0.7 mgMHETeq ·h-1·mgenzyme-1) and TPA (0.2 mgTPAeq ·h-1·mgenzyme-1) at 50 °C. TmFae-PETase showed a preference for the hydrolysis of ferulic acid methyl ester (MFA) in the presence of both PET and MFA. Site-directed mutagenesis and molecular dynamics simulations of TmFae-PETase revealed similar catalytic mechanisms for both PET and MFA. TmFae-PETase effectively depolymerized commercial PET, making it a promising candidate for application. Additionally, the known PET hydrolases IsPETase, FsC, and LCC also hydrolyzed MFA, indicating a potential origin of PET hydrolytic activity from its lignocellulosic-degrading abilities. This study provides an innovative strategy for screening PET-degrading enzymes identified from lignocellulose degradation-related enzymes within the gut microbiome of plastic-degrading mealworms. This discovery expands the existing pool of plastic-degrading enzymes available for resource recovery and bioremediation applications.

Monitoring corn stover processing by the fungus Ustilago maydis.

A key aspect of sustainable bioeconomy is the recirculation of renewable, agricultural waste streams as substrates for microbial production of high-value compounds. One approach is the bioconversion of corn stover, an abundant maize crop byproduct, using the fungal maize pathogen Ustilagomaydis. U.maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates. In this study, we demonstrate that U. maydis can grow using untreated corn stover as its sole carbon source. We developed a small-scale bioreactor platform to investigate U.maydis processing of corn stover, combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre- and post-fermentation residues. Our results reveal that U. maydis primarily utilizes soluble sugars i.e., glucose, sucrose and fructose present in corn stover, with only limited exploitation of the abundant lignocellulosic carbohydrates. Thus, we further explored the biotechnological potential of enhancing U.maydis´ lignocellulosic utilization. Additive performance improvements of up to 120% were achieved when using a maize mutant with increased biomass digestibility, co-fermentation with a commercial cellulolytic enzyme cocktail, and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes. This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U. maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates.

CRISPR/Cas9 mediated targeted knock-in of eglA gene to improve endoglucanase activity of Aspergillus fumigatus LMB-35Aa

Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.

Transcriptome Analysis of Ganoderma lingzhi Liquid Fermentation Process Using Corn Straw as Matrix

Ganoderma lingzhi, a species of white rot fungus, possesses the highest abundance of lignocellulose-degrading enzymes among these fungi, as well as a relatively high carbon conversion rate. Corn straw, as an important sustainable resource, is used as a substrate for the liquid culture of G. lingzhi. However, little is known about the genes encoding the lignocellulose degradation and polysaccharide and triterpenoid biosynthetic pathways involved in this process. This paper employs transcriptomics to uncover the key genes involved in lignocellulose degradation and the synthesis of polysaccharides and triterpenoids during the liquid fermentation of G. lingzhi using corn straw as the substrate, as well as their associations. Carbohydrate-Active enzymes analysis of differential genes in the sequencing results was used to analyze the genes related to lignocellulose degradation. Among these, 43 core genes encoding CAZymes were obtained after 0 to 5 days of fermentation, and 25 core genes encoding CAZymes were obtained after 5 to 12 days of fermentation. The differential expression levels of DN3690_c0_g1 (EGL), DN3627_c0_g2 (XYN), DN4778_c0_g1 (XYN), DN2037_c0_g1 (LACC), and DN277_c2_g1 (MnP) were used to identify the key genes. The polysaccharide synthesis metabolic pathway favored mannitol synthesis, and the expression of triterpene precursor-metabolizing enzyme genes revealed higher expression levels of key enzyme genes such as ACAT, HMGS, and MPK. A correlation clustering analysis of genes related to lignocellulose degradation, polysaccharide, and triterpene anabolism during liquid fermentation showed that lignocellulose degradation genes mainly influenced arabinose and mannitol anabolism, as well as the synthesis of triterpene precursors.

Screening, Production and Characterization of Potential Lignocellulolytic Actinomycetes from Agricultural Field

Actinomycetes are a suitable microbial group for the synthesis of lignocellulose-degrading enzymes. Enzymes that may degrade organic material, including cellulose, hemicellulose, and lignin, are released by actinomycetes. The aim of this research was to isolate actinomycetes from Rajkot, Gujarat, India’s soil and evaluate the activity of their cellulase and Xylanase enzymes. Starch Casein Agar (SCA) was used to identify a total of 30 isolates of actinomycetes. A qualitative plate assay (CMC-Na, Congo red) revealed that the highest zone of catalysis for MMD1 was 36 mm. Five strains were discovered to be effective for quantitative quantification of endoglucanase utilising filter paper and CMC as substrates: MMD1, MMD2, MMD3, MMD4, and MMD8. Following MMD 1 (endoglucanase 5.4 IU; FPase 4.4 IU), MMD 2 (endoglucanase 4.5 IU; FPase 3.4 IU) has demonstrated considerable endoglucanase and FPase activity. Beechwood xylan was used to treat sugarcane bagasse in order to test Xylanase, and 45% of the xylan (hemicellulose) fraction was obtained. MMD1 and MMD2 measured the xylanase enzyme activity (4.8IU and 4.2IU) in quantitative and qualitative assays (34 mm and 22 mm for BWX and 32 mm and 14 mm for agro-waste xylan). The strain MMD1 was identified as Streptomyces chartreusis through morphological, biochemical, and finally molecular characterization by 16S rRNA sequencing. It was then submitted to NCBI GenBank with the accession number MT254830.

Integrating multi-platform assembly to recover MAGs from hot spring biofilms: insights into microbial diversity, biofilm formation, and carbohydrate degradation

BackgroundHot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58–74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism.ResultsLeveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures.ConclusionsThis study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.

Alternative splicing analysis of lignocellulose-degrading enzyme genes and enzyme variants in Aspergillus niger

Alternative splicing (AS) greatly expands the protein diversity in eukaryotes. Although AS variants have been frequently reported existing in filamentous fungi, it remains unclear whether lignocellulose-degrading enzyme genes in industrially important fungi undergo AS events. In this work, AS events of lignocellulose-degrading enzymes genes in Aspergillus niger under two carbon sources (glucose and wheat straw) were investigated by RNA-Seq. The results showed that a total of 23 out of the 56 lignocellulose-degrading enzyme genes had AS events and intron retention was the main type of these AS events. The AS variant enzymes from the annotated endo-β-1,4-xylanase F1 gene (xynF1) and the endo-β-1,4-glucanase d gene (eglD), noted as XYNF1-AS and EGLD-AS, were characterized compared to their normal splicing products XYNF1 and EGLD, respectively. The AS variant XYNF1-AS displayed xylanase activity whereas XYNF1 did not. As for EGLD-AS and EGLD, neither of them showed annotated endo-β-1,4-glucanase activity. Instead, both showed lytic polysaccharide monooxygenase (LPMO) activity with some differences in catalytic properties. Our work demonstrated that the AS variants in A. niger were good sources for discovering novel lignocellulose-degrading enzymes.Key points• AS events were identified in the lignocellulose-degrading enzyme genes of A. niger.• New β-1,4-xylanase and LPMO derived from AS events were characterized.

An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.

The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA-sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA-sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. The tRNA-gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.

Microbial electrochemical composting: A sustainable strategy to enhance lignocellulose conversion into humus

Achieving satisfactory levels of humification during composting is recognised as a challenge due to the recalcitrance of lignocellulose and the uncertainty of current biofortification techniques. Therefore, a novel microbial electrochemical composting technology was developed from insights into microbiology and electrochemistry in this study to strengthen microorganisms’ function promoting the conversion from lignocellulose into humus. Firstly, a fungus (Aspergillus oryzae) was introduced which exhibited a higher propagation rate (29.9%) and secreted more lignocellulose-degrading enzymes (18.9–74.9%) under electric field assistance. Subsequently, an in-situ composting trial was conducted with supplementary electric fields simultaneously combined with Aspergillus oryzae inoculation. The abundance of Aspergillus oryzae increased 3.0–7.8-fold under the influence of electric field, which enabled the production of more extracellular enzymes (by 8.4–149.5%) to promote the conversion of lignocellulose to humus (increased by 7.0–20.2%). The results indicated electric field can act as an activator of Aspergillus oryzae and magnify its function. It provides an innovative and efficient technique for enhancing humification during composting that contributes to the conversion of organic waste into high-quality soil conditioner products.

<i>Nocardiopsis synnemataformans</i> NBRM9, an extremophilic actinomycete producing extremozyme cellulase, using lignocellulosic agro-wastes and its biotechnological applications

<abstract> <p>Actinomycetes are an attractive source of lignocellulose-degrading enzymes. The search for actinomycetes producing extremozyme cellulase using cheap lignocellulosic waste remains a priority goal of enzyme research. In this context, the extremophilic actinomycete NBRM9 showed promising cellulolytic activity in solid and liquid assays. This actinomycete was identified as <italic>Nocardiopsis synnemataformans</italic> based on its phenotypic characteristics alongside phylogenetic analyses of 16S rRNA gene sequencing (OQ380604.1). Using bean straw as the best agro-waste, the production of cellulase from this strain was statistically optimized using a response surface methodology, with the maximum activity (13.20 U/mL) achieved at an incubation temperature of 40 °C, a pH of 9, an incubation time of 7 days, and a 2% substrate concentration. The partially purified cellulase (PPC) showed promising activity and stability over a wide range of temperatures (20–90 °C), pH values (3–11), and NaCl concentrations (1–19%), with optimal activity at 50 °C, pH 9.0, and 10% salinity. Under these conditions, the enzyme retained >95% of its activity, thus indicating its extremozyme nature. The kinetics of cellulase showed that it has a V<sub>max</sub> of 20.19 ± 1.88 U/mL and a Km of 0.25 ± 0.07 mM. The immobilized PPC had a relative activity of 69.58 ± 0.13%. In the in vitro microtiter assay, the PPC was found to have a concentration-dependent anti-biofilm activity (up to 85.15 ± 1.60%). Additionally, the fermentative conversion of the hydrolyzed bean straw by <italic>Saccharomyces cerevisiae</italic> (KM504287.1) amounted to 65.80 ± 0.52% of the theoretical ethanol yield. Overall, for the first time, the present work reports the production of extremozymatic (thermo, alkali-, and halo-stable) cellulase from <italic>N. synnemataformans</italic> NBRM9. Therefore, this strain is recommended for use as a biotool in many lignocellulosic-based applications operating under harsh conditions.</p> </abstract>

Whole genome structural predictions reveal hidden diversity in putative oxidative enzymes of the lignocellulose-degrading ascomycete Parascedosporium putredinis NO1.

Economic valorization of lignocellulose is paramount to realizing a true circular bioeconomy; however, this requires the development of systems and processes to expand the repertoire of bioproducts beyond current renewable fuels, chemicals, and sustainable materials. Parascedosporium putredinis NO1 is an ascomycete that thrived at the later stages of a wheat-straw composting community culture, indicating a propensity to degrade recalcitrant lignin-enriched biomass, but exists within an underrepresented and underexplored fungal lineage. This strain has been proven to be an exciting candidate for the identification of new enzymes targeting recalcitrant components of lignocellulose following the recent discovery of a new lignin β-ether linkage cleaving enzyme. The first genome for the genus Parascedosporium for P. putredinis NO1 genome was sequenced, assembled, and annotated. The genome is 39 Mb in size, consisting of 21 contigs annotated to contain 9.998 protein-coding sequences. The carbohydrate-active enzyme (CAZyme) repertoire was compared to 2570 ascomycete genomes and in detail with Trichoderma reesei, Fusarium oxysporum, and sister taxa Scedosporium boydii. Significant expansion in the oxidative auxiliary activity class of CAZymes was observed in the P. putredinis NO1 genome, resulting from increased sequences encoding putative lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes acting within LPMO redox systems, and lignin-degrading laccases. P. putredinis NO1 scored above the 95th percentile for AA gene density across the ascomycete phylum, suggesting a primarily oxidative strategy for lignocellulose breakdown. Novel structure-based searching approaches were employed, revealing 17 new sequences with structural similarity to LPMO, laccase, and peroxidase sequences and which are potentially new lignocellulose-degrading enzymes.IMPORTANCEAn annotated reference genome has revealed P. putredinis NO1 as a useful resource for the identification of new lignocellulose-degrading enzymes for biorefining of woody plant biomass. Utilizing a "structure-omics"-based searching strategy, we identified new potentially lignocellulose-active sequences that would have been missed by traditional sequence searching methods. These new identifications, alongside the discovery of novel enzymatic functions from this underexplored lineage with the recent discovery of a new phenol oxidase that cleaves the main structural β-O-4 linkage in lignin from P. putredinis NO1, highlight the underexplored and poorly represented family Microascaceae as a particularly interesting candidate worthy of further exploration toward the valorization of high value biorenewable products.

Fecal Metagenomics Study Reveals That a Low-Fiber Diet Drives the Migration of Wild Asian Elephants in Xishuangbanna, China.

The rare northward migration of wild Asian elephants in Xishuangbanna, China, has attracted global attention. Elephant migration is a complex ecological process, and the factors driving this long-distance migration remain elusive. In this study, fresh fecal samples were collected from both captive and wild Asian elephants, along with breastfed calves residing within the Wild Elephant Valley of Xishuangbanna. Our aim was to investigate the relationship between diet, gut microbiota, and migration patterns in Asian elephants through comprehensive metagenomic sequencing analyses. Among the breastfed Asian elephant group, Bacteroidales and Escherichia emerged as the dominant bacterial taxa, while the primary carbohydrate-active enzymes (CAZymes) enriched in this group were GH2, GH20, GH92, GH97, GH38, GH23, and GH43, aligning with their dietary source, namely breast milk. The bacterial taxa enriched in captive Asian elephants (CAEs) were mainly Butyrivibrio, Treponema, and Fibrobacter, and the enriched lignocellulose-degrading enzymes mainly included GH25, GH10, GH9, and cellulase (EC 3.2.1.4). These findings are consistent with the high-fiber diet of captive elephants. In contrast, the main bacterial taxa enriched in wild Asian elephants (WAEs) were Ruminococcus and Eubacterium, and the enriched CAZymes included GH109, GH20, GH33, GH28, GH106, and GH39. The abundance of lignocellulose-degrading bacteria and CAZyme content was low in WAEs, indicating challenges in processing high-fiber foods and explaining the low-fiber diet in this group. These findings suggest that wild elephant herds migrate in search of nutritionally suitable, low-fiber food sources.

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.

Fungal Ligninolytic Enzymes and Their Application in Biomass Lignin Pretreatment.

Lignocellulosic biomass is a significant source of sustainable fuel and high-value chemical production. However, due to the complex cross-linked three-dimensional network structure, lignin is highly rigid to degradation. In natural environments, the degradation is performed by wood-rotting fungi. The process is slow, and thus, the use of lignin degradation by fungi has not been regarded as a feasible technology in the industrial lignocellulose treatment. Fungi produce a wide variety of ligninolytic enzymes that can be directly introduced in industrial processing of lignocellulose. Within this study, screening of ligninolytic enzyme production using decolorization of ABTS and Azure B dyes was performed for 10 fungal strains with potentially high enzyme production abilities. In addition to standard screening methods, media containing lignin and hay biomass as carbon sources were used to determine the change in enzyme production depending on the substrate. All selected fungi demonstrated the ability to adapt to a carbon source limitation; however, four strains indicated the ability to secrete ligninolytic enzymes in all experimental conditions-Irpex lacteus, Pleurotus dryinus, Bjerkandera adusta, and Trametes versicolor-respectively displayed a 100%, 82.7%, 82.7%, and 55% oxidation of ABTS on lignin-containing media and 100%, 87.9%, 78%, and 70% oxidation of ABTS on hay-containing media after 168 h of incubation. As a result, the most potent strains of fungi were selected to produce lignocellulose-degrading enzymes and to demonstrate their potential application in biological lignocellulose pretreatment.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

The renewable, abundant, and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples’ microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited β-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Key points• Sequence- and function-based metagenomics were used to find CAZymes in compost samples.• Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes.• Compost-derived fosmid libraries are enriched in clones with β-glucosidase activity.

Investigating the cellular functions of β-Glucosidases for synthesis of lignocellulose-degrading enzymes in Trichoderma reesei

β-glucosidases play an important role in the synthesis of cellulase in fungi, but their molecular functions and mechanisms remain unknown. We found that the 10 putative β-glucosidases investigated in Trichoderma reesei facilitate cellulase production, with cel3j being the most crucial. Transcriptional analysis revealed that the most affected biological processes in △cel3j strain were cellulase synthesis, ribosome biogenesis, and RNA polymerases. Moreover, CEL3J was unconventionally transported through the endoplasmic reticulum, bypassing the Golgi apparatus, whereas cel3j overexpression altered cellulase secretion from conventional to unconventional, likely owing to the activated unconventional protein secretion pathway (UPS), as indicated by the upregulation of genes related to UPS. The mTORC1-GRASP55 signaling axis may modulate the unconventional secretion of CEL3J and cellulase. The transcriptional levels of genes associated with DNA replication, the cell cycle, and meiosis were noticeably affected by overexpressing cel3j. These data give new clues for exploring the roles of β-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.

Lignin-degrading enzymes from a pathogenic canker-rot fungus Inonotus obliquus strain IO-B2

Inonotus obliquus is a pathogenic fungus found in living trees and has been widely used as a traditional medicine for cancer therapy. Although lignocellulose-degrading enzymes are involved in the early stages of host infection, the parasitic life cycle of this fungus has not been fully understood. In this study, we aimed to investigate the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) from I. obliquus cultivated in Kirk’s medium. The fungus was subjected to genome sequencing, and genes related to wood degradation were identified. The draft genome sequence of this fungus comprised 21,203 predicted protein-coding genes, of which 134 were estimated to be related to wood degradation. Among these, 47 genes associated with lignin degradation were found to have the highest number of mnp genes. Furthermore, we cloned the cDNA encoding a putative MnP, referred to as IoMnP1, and characterized its molecular structure. The results show that IoMnP1 has catalytic properties analogous to MnP. Phylogenetic analysis also confirmed that IoMnP1 was closely related to the MnPs from Pyrrhoderma noxium, Fomitiporia mediterranea, and Sanghuangporus baumii, which belong to the same family of Hymenochaetaceae. From the above results, we suggest that IoMnP1 is a member of MnPs.

Saccharification of sugarcane bagasse using cellulases and oxidoreductases enzymes

Lignin is the major polymer responsible for limiting the rates of lignocellulose degradation. Enzymatic hydrolysis of lignin (using oxidorreductases enzymes) open the way to action of cellulose and hemicellulose -degrading enzymes systems to obtain a fermentable hydrolysate rich in monomeric compounds from the carbohydrate polymers present in the biomass. Trichoderma reesei is an important fungus that is widely used as a source of cellulases for the hydrolysis of plant cell wall polysaccharides. In this study, was identified the filamentous fungi Pestalotiopsis sp BBF245, as a promising strain for production of oxidoreductases enzymes. Enzyme cocktails were composed, using cellulases from T. reesei and oxidoreductases enzymes from Pestalotiopsis sp BBF245 for hydrolysis of sugarcane bagasse (SCB) in natura. The amounts of reducing sugars released from the saccharification of bagasse was of 15,52 g/L after 24 hours of incubation. The results show that the combination of lignocellulose-degrading enzymes significantly increased the degradation of sugarcane bagasse and suggest that the use of enzyme cocktails oxidoreductases and cellulases may significantly improve the hydrolysis of biomass.

Site-directed modification of multifunctional lignocellulose-degrading enzymes of straw based on homologous modeling.

Studying the straw lignocellulose strengthening mechanism during simultaneous degradation has important practical significance for improving resource utilization and reducing environmental pollution. In this paper, the degradation ability of four straw lignocellulose-degrading enzymes was evaluated by molecular docking and molecular dynamics. Using the significantly binds to straw lignocellulose-degrading enzyme as a template, a multifunctional lignocellulose-degrading enzyme 3CBH-1KS5-4XQD-1B85 was constructed based on amino acid recombination and homologous modeling. Five efficient degrading enzymes (3CBH-1, 3CBH-2, 3CBH-3, 3CBH-4, and 3CBH-5) were designed by site-directed mutagenesis of 3CBH-1KS5-4XQD-1B85 amino acid at position 346. Molecular dynamics showed that the degradation ability of 3CBH-1 was significant and it was 1.45 times higher than 3CBH-1KS5-4XQD-1B85. Moreover, the mechanism of enhanced degradability and the stability of the enzymes were explored. With the aid of Taguchi experiments, the suitable external environment for degrading straw was determined. In the presence of inhibitors (organic acids and phenolic compounds), the binding energy of 3CBH-1 (238.46 ± 30.96kJ/mol) is 36.42% higher than that of 3CBH-1KS5-4XQD-1B85 (174.79 ± 20.35kJ/mol) without external environmental stimulation. Based on homology modeling, this paper constructed a site-directed mutagenesis scheme of multifunctional enzymes, and the aim was to obtain multifunctional and efficient straw lignocellulose-degrading enzymes through protein engineering, which provided a feasible scheme for straw biodegradation.