Microbiota succession influences nematode physiology in a beetle microcosm ecosystem.

Unravelling the multifaceted and bidirectional interactions between microbiota and host physiology represents a major scientific challenge. Here, we utilise the nematode model, Pristionchus pacificus, coupled to a laboratory-simulated decay process of its insect host, to mimic natural microbiota succession and investigate associated tripartite interactions. Metagenomics reveal that during initial decay stages, the population of vitamin B-producing bacteria diminishes, potentially due to a preferential selection by nematodes. As decay progresses to nutrient-depleted stages, bacteria with smaller genomes producing less nutrients become more prevalent. Lipid utilisation and dauer formation, representing key nematode survival strategies, are influenced by microbiota changes. Additionally, horizontally acquired cellulases extend the nematodes' reproductive phase due to more efficient foraging. Lastly, the expressions of Pristionchus species-specific genes are more responsive to natural microbiota compared to conserved genes, suggesting their importance in the organisms' adaptation to its ecological niche. In summary, we show the importance of microbial successions and their reciprocal interaction with nematodes for insect decay in semi-artificial ecosystems.

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 100 g/L and enzyme dosage of 4 mg/g, glucose of 17.6 g/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.

Unveiling a Microexon Switch: Novel Regulation of the Activities of Sugar Assimilation and Plant-Cell-Wall-Degrading Xylanases and Cellulases by Xlr2 in Trichoderma virens.

Functional microexons have not previously been described in filamentous fungi. Here, we describe a novel mechanism of transcriptional regulation in Trichoderma requiring the inclusion of a microexon from the Xlr2 gene. In low-glucose environments, a long mRNA including the microexon encodes a protein with a GAL4-like DNA-binding domain (Xlr2-α), whereas in high-glucose environments, a short mRNA that is produced encodes a protein lacking this DNA-binding domain (Xlr2-ß). Interestingly, the protein isoforms differ in their impact on cellulase and xylanase activity. Deleting the Xlr2 gene reduced both xylanase and cellulase activity and growth on different carbon sources, such as carboxymethylcellulose, xylan, glucose, and arabinose. The overexpression of either Xlr2-α or Xlr2-ß in T. virens showed that the short isoform (Xlr2-ß) caused higher xylanase activity than the wild types or the long isoform (Xlr2-α). Conversely, cellulase activity did not increase when overexpressing Xlr2-ß but was increased with the overexpression of Xlr2-α. This is the first report of a novel transcriptional regulation mechanism of plant-cell-wall-degrading enzyme activity in T. virens. This involves the differential expression of a microexon from a gene encoding a transcriptional regulator.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates.

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and ß-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Parasitic dodder expresses an arsenal of secreted cellulases with multi-substrate specificity during host invasion.

Cuscuta campestris is a common and problematic parasitic plant which relies on haustoria to connect to and siphon nutrients from host plants. Glycoside hydrolase family 9 (GH9) cellulases (EC 3.2.1.4) play critical roles in plant cell wall biosynthesis and disassembly, but their roles during Cuscuta host invasion remains underexplored. In this study, we identified 22 full-length GH9 cellulase genes in C. campestris genome, which encoded fifteen secreted and seven membrane-anchored cellulases that showed distinct phylogenetic relationships. Expression profiles suggested that some of the genes are involved in biosynthesis and remodeling of the parasite's cell wall during haustoriogenesis, while other genes encoding secreted B- and C-type cellulases are tentatively associated with degrading host cell walls during invasion. Transcriptomic data in a host-free system and in the presence of susceptible or partially resistant tomato hosts, showed for especially GH9B7, GH9B11 and GH9B12 a shift in expression profiles in the presence of hosts, being more highly expressed during host attachment, indicating that Cuscuta can tune cellulase expression in response to a host. Functional analyses of recombinant B- and C-type cellulases showed endoglucanase activities over wide pH and temperature conditions, and activities towards multiple cellulose and hemicellulose substrates. These findings improve our understanding of host cell wall disassembly by Cuscuta, and cellulase activity towards broad substrate range potentially explain its wide host range. This is the first study to provide a broad biochemical insight into Cuscuta GH9 cellulases, which based on our study may have potential applications in industrial bioprocessing.

The role of the Flb protein family in the life cycle of Aspergillus niger.

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from a Trichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolase II. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

Enhancement of cellulase production by cellulolytic bacteria SB125 in submerged fermentation medium and biochemical characterization of the enzyme.

Microbial diversity from indigenous cultures has the potential to accelerate lignocellulose degradation through enzymes and make composting economically feasible. Therefore, this study is designed to boost cellulase output from a bacterial strain obtained from soil using a one-variable-at-a-time approach and response surface methodology. The bacteria recognized as Bacillus tequilensis (ON754229) produced the maximum cellulase at a temperature of 37 °C, pH -7.0, and incubation time of 72 h. A major contribution was anticipated by glucose (17 %) and ammonium sulfate (11 %) with cellulase activity of 0.56 U/mL in the optimized medium. The enzyme possessed activity of CMCase, FPase, and amylase of 0.589 µmol/min, 1.22 µmol/min, and 0.92 µmol/min respectively. SDS-PAGE showed a 65 kDa molecular weight of the enzyme capable of degrading cellulose, as confirmed by zymogram analysis. The enzyme showed relatively moderate thermo-stability towards neutral pH conditions possessing optimum conditions at pH 6.5 and temperature of 50 °C. The Km and Vmax values were 11.44 mM and 0.643 µmol/min respectively. The presence of MgSO4, ZnSO4, and Triton X- 100 increased the enzymatic reaction however AgNO3, EDTA, and HgCl2 altered the activation process. These results showed cellulase from B. tequilensis SB125 would be suitable for conventional industrial processes that convert biomass into biofuels.

Analysis of endoglucanases production using metatranscriptomics and proteomics approach.

The cellulases are among the most used enzyme in industries for various purposes. They add up to the green economy perspective and cost-effective production of enterprises. Biorefineries, paper industries, and textile industries are foremost in their usage. The production of endoglucanases from microorganisms is a valuable resource and can be exploited with the help of biotechnology. The present review provides some insight into the uses of endoglucanases in different industries and the potent fungal source of these enzymes. The advances in the enzyme technology has helped towards understanding some pathways to increase the production of industrial enzymes from microorganisms. The proteomics analysis and systems biology tools also help to identify these pathways for the enhanced production of such enzymes. This review deciphers the use of proteomics tools to analyze the potent microorganisms and identify suitable culture conditions to increase the output of endoglucanases. The review also includes the role of quantitative proteomics which is a powerful technique to get results faster and more timely. The role of metatranscriptomic approaches are also described which are helpful in the enzyme engineering for their efficient use under industrial conditions. Conclusively, this review helps to understand the challenges faced in the industrial use of endoglucanases and their further improvement.

Glycosylation of cellulase: a novel strategy for improving cellulase.

Protein glycosylation is the most complex posttranslational modification process. Most cellulases from filamentous fungi contain N-glycosylation and O-glycosylation. Here, we discuss the potential roles of glycosylation on the characteristics and function of cellulases. The use of certain cultivation, inducer, and alteration of engineering glycosylation pathway can enable the rational control of cellulase glycosylation. Glycosylation does not occur arbitrarily and may tend to modify the 3D structure of cellulases by using specially distributed glycans. Therefore, glycoengineering should be considered comprehensively along with the spatial structure of cellulases. Cellulase glycosylation may be an evolution phenomenon, which has been considered as an economical way for providing different functions from identical proteins. In addition to gene and transcription regulations, glycosylation may be another regulation on the protein expression level. Enhanced understanding of the potential regulatory role of cellulase glycosylation will enable synthetic biology approaches for the development of commercial cellulase.

Long noncoding RNA TRABA suppresses ß-glucosidase-encoding BGLU24 to promote salt tolerance in cotton.

Salt stress severely damages the growth and yield of crops. Recently, long noncoding RNAs (lncRNAs) were demonstrated to regulate various biological processes and responses to environmental stresses. However, the regulatory mechanisms of lncRNAs in cotton (Gossypium hirsutum) response to salt stress are still poorly understood. Here, we observed that a lncRNA, trans acting of BGLU24 by lncRNA (TRABA), was highly expressed while GhBGLU24-A was weakly expressed in a salt-tolerant cotton accession (DM37) compared to a salt-sensitive accession (TM-1). Using TRABA as an effector and proGhBGLU24-A-driven GUS as a reporter, we showed that TRABA suppressed GhBGLU24-A promoter activity in double transgenic Arabidopsis (Arabidopsis thaliana), which explained why GhBGLU24-A was weakly expressed in the salt-tolerant accession compared to the salt-sensitive accession. GhBGLU24-A encodes an endoplasmic reticulum (ER)-localized ß-glucosidase that responds to salt stress. Further investigation revealed that GhBGLU24-A interacted with RING-type E3 ubiquitin ligase (GhRUBL). Virus-induced gene silencing (VIGS) and transgenic Arabidopsis studies revealed that both GhBGLU24-A and GhRUBL diminish plant tolerance to salt stress and ER stress. Based on its substantial effect on ER-related degradation (ERAD)-associated gene expression, GhBGLU24-A mediates ER stress likely through the ERAD pathway. These findings provide insights into the regulatory role of the lncRNA TRABA in modulating salt and ER stresses in cotton and have potential implications for developing more resilient crops.

Transcriptome and photosynthetic analyses provide new insight into the molecular mechanisms underlying heat stress tolerance in Rhododendron × pulchrum Sweet.

Rhododendron species provide excellent ornamental use worldwide, yet heat stress (HS) is one of the major threats to their cultivation. However, the intricate mechanisms underlying the photochemical and transcriptional regulations associated with the heat stress response in Rhododendron remain relatively unexplored. In this study, the analyses of morphological characteristics and chlorophyll fluorescence (ChlF) kinetics showed that HS (40 °C/35 °C) had a notable impact on both the donor's and acceptor's sides of photosystem II (PSII), resulting in reduced PSII activity and electron transfer capacity. The gradual recovery of plants observed following a 5-day period of culture under normal conditions indicates the reversible nature of the HS impact on Rhododendron × pulchrum. Analysis of transcriptome data unveiled noteworthy trends: four genes associated with photosynthesis-antenna protein synthesis (LHCb1, LHCb2 and LHCb3) and the antioxidant system (glutamate-cysteine ligase) experienced significant down-regulation in the leaves of R. × pulchrum during HS. Conversely, aseorbate peroxidase and glutathione S-transferase TAU 8 demonstrated an up-regulated pattern. Furthermore, six down-regulated genes (phos-phoenolpyruvate carboxylase 4, sedoheptulose-bisphosphatase, ribose-5-phosphate isomerase 2, high cyclic electron flow 1, beta glucosidase 32 and starch synthase 2) and two up-regulated genes (beta glucosidase 2 and UDP-glucose pyrophosphorylase 2) implicated in photosynthetic carbon fixation and starch/sucrose metabolism were identified during the recovery process. To augment these insights, a weighted gene co-expression network analysis yielded a co-expression network, pinpointing the hub genes correlated with ChlF dynamics' variation trends. The cumulative results showed that HS inhibited the synthesis of photosynthesis-antenna proteins in R. × pulchrum leaves. This disruption subsequently led to diminished photochemical activities in both PSII and PSI, albeit with PSI exhibiting heightened thermostability. Depending on the regulation of the reactive oxygen species scavenging system and heat dissipation, photoprotection sustained the recoverability of R. × pulchrum to HS.

Potential of psychrotolerant rhizobacteria for the growth promotion of wheat (Triticum aestivum L.).

Wheat is the second most important staple crop grown and consumed worldwide. Temperature fluctuations especially the cold stress during the winter season reduces wheat growth and grain yield. Psychrotolerant plant growth-promoting rhizobacteria (PGPR) may improve plant stress-tolerance in addition to serve as biofertilizer. The present study aimed to isolate and identify PGPR, with the potential to tolerate cold stress for subsequent use in supporting wheat growth under cold stress. Ten psychrotolerant bacteria were isolated from the wheat rhizosphere at 4 °C and tested for their ability to grow at wide range of temperature ranging from -8 °C to 36 °C and multiple plant beneficial traits. All bacteria were able to grow at 4 °C to 32 °C temperature range and solubilized phosphorus except WR23 at 4 °C, whereas all the bacteria solubilized phosphorus at 28 °C. Seven bacteria produced indole-3-acetic acid at 4 °C, whereas all produced indole-3-acetic acid at 28 °C. Seven bacteria showed the ability to fix nitrogen at 4 °C, while all the bacteria fixed nitrogen at 28 °C. Only one bacterium showed the potential to produce cellulase at 4 °C, whereas four bacteria showed the potential to produce cellulase at 28 °C. Seven bacteria produced pectinase at 4 °C, while one bacterium produced pectinase at 28 °C. Only one bacterium solubilized the zinc at 4 °C, whereas six bacteria solubilized the zinc at 28 °C using ZnO as the primary zinc source. Five bacteria solubilized the zinc at 4 °C, while seven bacteria solubilized the zinc at 28 °C using ZnCO3 as the primary zinc source. All the bacteria produced biofilm at 4 °C and 28 °C. In general, we noticed behavior of higher production of plant growth-promoting substances at 28 °C, except pectinase assay. Overall, in vitro testing confirms that microbes perform their inherent properties efficiently at optimum temperatures rather than the low temperatures due to high metabolic rate. Five potential rhizobacteria were selected based on the in vitro testing and evaluated for plant growth-promoting potential on wheat under controlled conditions. WR22 and WR24 significantly improved wheat growth, specifically increasing plant dry weight by 42% and 58%, respectively. 16S rRNA sequence analysis of WR22 showed 99.78% similarity with Cupriavidus campinensis and WR24 showed 99.9% similarity with Enterobacter ludwigii. This is the first report highlighting the association of C. campinensis and E. ludwigii with wheat rhizosphere. These bacteria can serve as potential candidates for biofertilizer to mitigate the chilling effect and improve wheat production after field-testing.

Oriented display of cello-oligosaccharides for pull-down binding assays to distinguish binding preferences of glycan binding proteins.

The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to a sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To better understand the binding of type B CBMs towards smaller cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This may indicate a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.

Preliminary study on the anti-CO2 stress and growth ability of hypsizygus marmoreus mutant strain HY68.

BACKGROUND: A high concentration of CO2 will stagnate the development of the newly formed primordia of Hypsizygus marmoreus, hinder the development of the mushroom cap, thereby inhibiting the normal differentiation of the fruiting body. Moreover, in the previous experiment, our research group obtained the mutant strain HY68 of H. marmoreus, which can maintain normal fruiting under the condition of high concentration of CO2. Our study aimed to evaluate the CO2 tolerance ability of the mutant strain HY68, in comparison with the starting strain HY61 and the control strain HY62. We analyzed the mycelial growth of these strains under various conditions, including different temperatures, pH levels, carbon sources, and nitrogen sources, and measured the activity of the cellulose enzyme. Additionally, we identified and predicted ß-glucosidase-related genes in HY68 and analyzed their gene and protein structures. RESULTS: Our results indicate that HY68 showed superior CO2 tolerance compared to the other strains tested, with an optimal growth temperature of 25 °C and pH of 7, and maltose and beef paste as the ideal carbon and nitrogen sources, respectively. Enzyme activity assays revealed a positive correlation between ß-glucosidase activity and CO2 tolerance, with Gene14147 identified as the most closely related gene to this activity. Inbred strains of HY68 showed trait segregation for CO2 tolerance. CONCLUSIONS: Both HY68 and its self-bred offspring could tolerate CO2 stress. The fruiting period of the strains resistant to CO2 stress was shorter than that of the strains not tolerant to CO2 stress. The activity of ß-GC and the ability to tolerate CO2 were more closely related to the growth efficiency of fruiting bodies. This study lays the foundation for understanding how CO2 regulates the growth of edible fungi, which is conducive to the innovation of edible fungus breeding methods. The application of the new strain HY68 is beneficial to the research of energy-saving production in factory cultivation.

Production of fermentable glucose from bioconversion of cellulose using efficient microbial cellulases produced from water hyacinth waste.

The economic production of cellulase enzymes for various industrial applications is one of the major research areas. A number of broad industrial applications, for example, in cellulosic biomass hydrolysis for simple sugars such as glucose and subsequent biofuel production, make these enzyme systems the third most demanding enzymes. Nevertheless, due to their production on commercial substrates, cellulases fall into the category of costly enzymes. Therefore, the goal of the present work is to evaluate the enhancement of cellulase production and its utilization in the enzymatic hydrolysis of biomass using low-cost cellulosic substrate, which is abundant and widely available. In this context, waste biomasses of water hyacinth (WH), including leaves and stems, have been used as feedstock to produce cellulases via solid-state fermentation (SSF) in the current study, which improves its production as well as activity. Furthermore, the impact of process parameters like temperature and pH has been investigated for improved cellulase production. At optimum concentration using 10 g of feedstock, 22 IU/gds of FP, 92 IU/gds of BGL, and 111 IU/gds of EG have been noticed in day 5 of SSF. Herein, 40 °C has been identified as the optimum temperature for cellulase production, whereas 50-55 °C has been recorded as the optimum reaction temperature for cellulase enzyme activity. Additionally, pH 5.5 has been identified as the optimum pH for cellulase enzyme production, whereas this enzyme was thermally stable (55 °C) at pH 5.0 up to 3.5 h. Further, the cellulosic biomass hydrolysis of WH leaves via an optimized crude enzyme has been performed, and this could release 24.34 g/L of glucose in 24 h of the reaction. The current findings may have potential for developing cellulases for mass-scale production using WH-based waste bioresources for numerous biorefinery applications.

Discovery of two bifunctional/multifunctional cellulases by functional metagenomics.

Cellulases are widely used in industry, and the usage in bioconversion of biofuels makes cellulases more valuable. In this study, two tandem genes that encoded cellulases ZF994-1 and ZF994-2, respectively, were identified on a cosmid from a soil metagenomic library. Phylogenetic analysis indicated that ZF994-1 and ZF994-2 belonged to glycoside hydrolase family 12 (GH12), and GH3, respectively. Based on the substrate specificity analysis, the recombinant ZF994-1 exhibited weak endoglucanase activity, moderate ß-1,3-glucanase and ß-1,4-mannanase activities, and strong ß-glucosidase activity, while the recombinant ZF994-2 exhibited moderate endoglucanase activity and strong ß-glucosidase activity. More than 45% ß-glucosidase activity of the recombinant ZF994-1 retained in the buffer containing 3 M glucose, indicating the good tolerance against glucose. The recombinant ZF994-2 showed high activity in the presence of metal ions and organic reagents, exhibiting potential industrial applications.

Draft genome sequence of Hahella sp. CR1 and its ability in producing cellulases for saccharifying agricultural biomass.

Hahella is a genus that has not been well-studied, with only two identified species. The potential of this genus to produce cellulases is yet to be fully explored. The present study isolated Hahella sp. CR1 from mangrove soil in Tanjung Piai National Park, Malaysia, and performed whole genome sequencing (WGS) using NovaSeq 6000. The final assembled genome consists of 62 contigs, 7,106,771 bp, a GC ratio of 53.5%, and encoded for 6,397 genes. The CR1 strain exhibited the highest similarity with Hahella sp. HN01 compared to other available genomes, where the ANI, dDDH, AAI, and POCP were 97.04%, 75.2%, 97.95%, and 91.0%, respectively. In addition, the CAZymes analysis identified 88 GTs, 54 GHs, 11 CEs, 7 AAs, 2 PLs, and 48 CBMs in the genome of strain CR1. Among these proteins, 11 are related to cellulose degradation. The cellulases produced from strain CR1 were characterized and demonstrated optimal activity at 60 ℃, pH 7.0, and 15% (w/v) sodium chloride. The enzyme was activated by K+, Fe2+, Mg2+, Co2+, and Tween 40. Furthermore, cellulases from strain CR1 improved the saccharification efficiency of a commercial cellulase blend on the tested agricultural wastes, including empty fruit bunch, coconut husk, and sugarcane bagasse. This study provides new insights into the cellulases produced by strain CR1 and their potential to be used in lignocellulosic biomass pre-treatment.

Different techniques reveal the difference of community structure and function of fungi from root and rhizosphere of Salvia miltiorrhiza Bunge.

Fungi have essential functions in plant health and performance. However, the plant-associated functions of many cultured fungi have not been established in detail. Here, the fungal species diversity in Salvia miltiorrhiza roots and rhizosphere was assessed for the first time using culturomics and high-throughput sequencing. We present a comprehensive functional metagenomic analysis of these fungi and verified activity of cellulase and chitinase predicted in the metagenomic analysis. We first collected and cultured fungi from the root and rhizosphere of S. miltiorrhiza. We found 92 species across 37 families and five phyla, with Ascomycota being dominant. Many rDNA internal transcribed spacer sequences could not be assigned to lower taxonomic levels. There were 19 genera of endophytic fungi and 37 genera of rhizosphere fungi. The culturomics approach had lower taxonomic diversity than high-throughput sequencing, but some fungi were only found in cultures. Structural analyses indicated that the dominant species differed in cultured and non-cultured samples at other levels, apart from the phylum level. Functional analysis mapped 223 carbohydrate enzyme families and 393 pathways in the CAZy and KEGG databases, respectively. The most abundant families were glycoside hydrolases and those involved in carbohydrate metabolism. As predicted by metagenomics, we experimentally verified cellulase and chitinase activity for 29 and 74 fungi, respectively. We provide the first evidence of biomass recycling by fungi that are associated with plants. Culturing is essential to reveal the hidden microbial community and critical functions in plant-microbe interactions.

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 ß-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 ß-glucosidase activity.