Plant Biomass Deconstruction Research Articles

Improved Cellulase Production of Trichoderma reesei by Regulating Mycelium Morphology

The small GTPases of the Rho family are known to regulate various biological processes in filamentous fungi. In this study, we investigated the impact of deleting Rho proteins on the growth and cellulase production of Trichoderma reesei. Our findings revealed that deletion of cdc42 led to the most severe growth defect and impaired cellulase production. Conversely, overexpression of cdc42 resulted in a hyperbranched phenotype, significantly enhancing cellulase production. Furthermore, the cdc42-overexpressing (OCdc42) strain showed an increased expression of multiple cellulase genes and Rho GTPase genes. Analysis of the secretome in the OCdc42 strain unveiled an increased abundance and diversity of extracellular proteins compared to the parent strain. These discoveries provide valuable insights into the functionality of Rho GTPases in T. reesei and offer potential targets for engineering fungi to improve plant biomass deconstruction in biorefineries.

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.

Advances in microbial pretreatment for biorefining of perennial grasses.

Perennial grasses are potentially abundant sources of biomass for biorefineries, which can produce high yields with low input requirements, and many added environmental benefits. However, perennial grasses are highly recalcitrant to biodegradation and may require pretreatment before undergoing many biorefining pathways. Microbial pretreatment uses the ability of microorganisms or their enzymes to deconstruct plant biomass and enhance its biodegradability. This process can enhance the enzymatic digestibility of perennial grasses, enabling saccharification with cellulolytic enzymes to produce fermentable sugars and derived fermentation products. Similarly, microbial pretreatment can increase the methanation rate when the grasses are used to produce biogas through anaerobic digestion. Microorganisms can also increase the digestibility of the grasses to improve their quality as animal feed, enhance the properties of grass pellets, and improve biomass thermochemical conversion. Metabolites produced by fungi or bacteria during microbial pretreatment, such as ligninolytic and cellulolytic enzymes, can be further recovered as added-value products. Additionally, the action of the microorganisms can release chemicals with commercialization potential, such as hydroxycinnamic acids and oligosaccharides, from the grasses. This review explores the recent advances and remaining challenges in using microbial pretreatment for perennial grasses with the goal of obtaining added-value products through biorefining. It emphasizes recent trends in microbial pretreatment such as the use of microorganisms as part of microbial consortia or in unsterilized systems, the use and development of microorganisms and consortia capable of performing more than one biorefining step, and the use of cell-free systems based on microbial enzymes. KEY POINTS: • Microorganisms or enzymes can reduce the recalcitrance of grasses for biorefining • Microbial pretreatment effectiveness depends on the grass-microbe interaction • Microbial pretreatment can generate value added co-products to enhance feasibility.

Carbohydrate Depolymerization by Intricate Cellulosomal Systems.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

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.

Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction.

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

Characterizing the Alteration in Rumen Microbiome and Carbohydrate-Active Enzymes Profile with Forage of Muskoxen Rumen through Comparative Metatranscriptomics.

Muskox (Ovibos moschatus), as the biggest herbivore in the High Arctic, has been enduring the austere arctic nutritional conditions and has evolved to ingest and digest scarce and high lignified forages to support the growth and reproduce, implying probably harbor a distinct microbial reservoir for the deconstruction of plant biomass. Therefore, metagenomics approach was applied to characterize the rumen microbial community and understand the alteration in rumen microbiome of muskoxen fed either triticale straw or brome hay. The difference in the structure of microbial communities including bacteria, archaea, fungi, and protozoa between the two forages was observed at the taxonomic level of genus. Further, although the highly abundant phylotypes in muskoxen rumen fed either triticale straw or brome hay were almost the same, the selective enrichment different phylotypes for fiber degrading, soluble substrates fermenting, electron and hydrogen scavenging through methanogenesis, acetogenesis, propionogenesis, and sulfur-reducing was also noticed. Specifically, triticale straw with higher content of fiber, cellulose selectively enriched more lignocellulolytic taxa and electron transferring taxa, while brome hay with higher nitrogen content selectively enriched more families and genera for degradable substrates-digesting. Intriguingly, the carbohydrate-active enzyme profile suggested an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on triticale straw. The majority of the cellulases belonged to fiver GH families (i.e., GH5, GH6, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus, Piromyces, Neocallimastix, and Fibrobacter. Abundance of major genes coding for hemicellulose digestion was higher than cellulose mainly including GH8, GH10, GH16, GH26, and GH30, and these enzymes were produced by members of the genera Fibrobacter, Ruminococcus, and Clostridium. Oligosaccharides were mainly of the GH1, GH2, GH3, and GH31 types and were associated with the genera Prevotella and Piromyces. Our results strengthen metatranscriptomic evidence in support of the understanding of the microbial community and plant polysaccharide response to changes in the feed type and host animal. The study also establishes these specific microbial consortia procured from triticale straw group can be used further for efficient plant biomass hydrolysis.

Thermoxylanolytic and Thermosaccharolytic Potential of a Heat Adapted Bacterial Consortium Developed From Goat Rumen Contents

Thermoactive xylanases have important applications in the industrial deconstruction of lignocellulosic plant biomass, due to their sustained activity even at high temperature conditions of industrial bioreactors. We herein report the development of a thermoactive xylanolytic microbial consortium from the semi-digested contents of goat rumen and characterization of the xylanolytic enzyme cocktail produced by it. The consortium exhibited maximum endoxylanase activity at pH6 and at 60°C. Zymogram analysis revealed the production of multiple xylanases. The xylanase cocktail was stable over a pH range of 5–9 after pre-incubation for 3 h. It retained 74% activity after pre-incubation (60°C) for 50 min. It’s activity was enhanced in presence of β-mercaptoethanol, NH4+, Mg2⁺ and Ca2⁺, whereas Hg2⁺ had an inhibitory effect. The xylanolytic cocktail was further utilized for the saccharification of alkali pre-treated rice straw and mushroom spent rice straw. Saccharification was studied quantitatively using the dinitrosalicylic acid method and qualitatively using scanning electron microscopy. Results indicated the potential of the xylanolytic cocktail for the saccharification of rice straw and highlighted the significance of chemical and/or biological pre-treatment in improving the accessibility of the substrate towards the xylanase cocktail.

Coexpression of a β-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Functional screening of a Caatinga goat (Capra hircus) rumen metagenomic library reveals a novel GH3 β-xylosidase.

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol.

Structural and molecular dynamics investigations of ligand stabilization via secondary binding site interactions in Paenibacillus xylanivorans GH11 xylanase

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-β-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

Flowthrough Pretreatment of Softwood under Water-Only and Alkali Conditions

Flowthrough pretreatment provides valuable insight into the fundamentals of deconstruction of plant biomass. In this study, the potential softwood degradation pathways under water-only and alkali c...

Tibetan Sheep Adapt to Plant Phenology in Alpine Meadows by Changing Rumen Microbial Community Structure and Function.

The rumen microbiota is strongly associated with host health, nutrient absorption, and adaptability. However, the composition, functioning and adaptability of rumen microbiota in Tibetan sheep (TS) across different phenological periods are unclear. In this study we used sequencing of the V4-V5 region of 16S rRNA, qPCR technology and metagenomics to investigate the adaption of rumen microbiota to forage in different stages of phenology. In a grassy period, due to the high nutritional quality of the forage, TS can produce high concentrations of NH3-N and short fatty acids by increasing the content of key bacteria in the rumen, such as Bacteroidetes, Prevotella, Succiniclasticum, Treponema, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens to aid in growth. In the withering period, there was a positive correlation between microorganisms which indicated the closely cooperation between microorganisms, and metagenomic analysis showed that the high genes (GHs and CBMs) and subtribe (GH8, GH12, GH45, GH6, GH9, GH5, GH10, GH3, GH52, GH11, GH57, CBM1, CBM4, CBM6, CBM16, CBM37, CBM13, CBM35, CBM42, CBM32, and CBM62) that encode cellulolytic enzymes were significantly increased when the host faced low quantity and quality of forage. Genes involved in metabolic pathways, fatty acid biosynthesis and biosynthesis of antibiotics were significantly enriched, which indicated that rumen microbiota could improve plant biomass deconstruction and energy maintenance in the face of nutritional deficiencies. In the regreen period, both the composition and function of rumen microbiota had obvious disadvantages, therefore, to improve the competitiveness of microorganisms, we suggest TS should be supplemented with high-protein feed. This study is of great significance for exploring the high altitude adaptability of TS.

Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to β-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60°C and is thermo-resistance at 65°C. XynA maintains 66% of the enzymatic activity at high temperatures (90°C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50μmolmL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78μmolmL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched forsuch bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded β-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Understanding the alteration in rumen microbiome and CAZymes profile with diet and host through comparative metagenomic approach.

Rumen microbial community harbors a distinct genetic reservoir of potent carbohydrate-active enzymes (CAZyme) that functions efficiently for the deconstruction of plant biomass. Based on this premise, metagenomics approach was applied to characterize the rumen microbial community and identify carbohydrate-active genes of Bos taurus (cow) and Bubalus bubalis (buffalo) fed on green or dry roughage. Metadata was generated from the samples: green roughage-fed cow (NDC_GR), buffalo (NDB_GR) and dry roughage-fed cow (NDC_DR), buffalo (NDB_DR). Phylogenetic analysis revealed the dominance of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and Fibrobacter in all the four samples, covering 90-96% of the total bacterial population. On finer resolution, higher abundance of bacterial genera Fibrobacter, Bacteroides, Clostridium, Prevotella and Ruminococcus involved in plant biomass hydrolysis was observed in NDB_DR. Functional annotation using dbCAN annotation algorithm identified 28.13%, 8.08% 10.93% and 12.53% of the total contigs as putatively carbohydrate-active against NDC_GR, NDB_GR, NDC_DR and NDB_DR, respectively. Additional profiling of CAZymes revealed an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on dry roughage with substantial enrichments of genes encoding GHs from families GH2, GH3, GH13 and GH43. GHs of families GH45, GH12, GH113, GH128, GH54 and GH27 were observed exclusively in NDB_DR metagenome. A higher abundance of cellulases, hemicellulases, debranching and oligosaccharide hydrolyzing enzymes was revealed in NDB_DR metagenome. Accordingly, it can be concluded that buffalo rumen microbiome are more efficient in plant biomass hydrolysis. The present study provides a deep understanding of the shifts in microbial community and plant polysaccharide deconstructing capabilities of rumen microbiome in response to changes in the feed type and host animal. Activity-specific microbial consortia procured from these animals can be used further for efficient plant biomass hydrolysis. The study also establishes the utility of rumen microbiome as a unique resource for mining diverse lignocellulolytic enzymes.

A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei

Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

Imaging Changes in Cell Walls of Engineered Poplar by Stimulated Raman Scattering and Atomic Force Microscopy

Lignin is a major impediment in the deconstruction of plant biomass to its soluble monomeric constituents. Modification of the lignin biosynthetic pathway has proven to be an effective means of red...