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.

Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, ß-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled ß-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled ß-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

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.

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.

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.

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.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

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.

Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus.

Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce enzymes that metabolize this polymer. In this work we have demonstrated the presence of cellulolytic activity in the supernatant of Scenedesmus quadricauda cultures and we identified the presence of extracellular cellulases in the genome of five Scenedesmus species. Scenedesmus is a green alga which grows in both freshwater and saltwater regions as well as in soils, showing highly flexible metabolic properties. Sequence comparison of the different identified cellulases with hydrolytic enzymes from other organisms using multisequence alignments and phylogenetic trees showed that these proteins belong to the families of glycosyl hydrolases 1, 5, 9, and 10. In addition, most of the Scenedesmus cellulases showed greater sequence similarity with those from invertebrates, fungi, bacteria, and other microalgae than with the plant homologs. Furthermore, the data obtained from the three dimensional structure showed that both, their global structure and the main amino acid residues involved in catalysis and substrate binding are well conserved. Based on our results, we propose that different species of Scenedesmus could act as biocatalysts for the hydrolysis of cellulosic biomass produced from sunlight.

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.

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.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Non-redundant functionality of Lactiplantibacillus plantarum phospho-ß-glucosidases revealed by carbohydrate utilization signatures associated to pbg2 and pbg4 gene mutants.

AIM: To increase our knowledge on the functionality of 6-phospho-ß-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. METHODS AND RESULTS: Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-ß-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild-type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. CONCLUSION: Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-ß-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.

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.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable ß-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable ß-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

Sucrose-nonfermenting 1 kinase activates histone acetylase GCN5 to promote cellulase production in Trichoderma.

Trichoderma serves as the primary producer of cellulases and hemicellulases in industrial settings as it readily secretes a variety of cellulolytic enzymes. The protein kinase SNF1 (sucrose-nonfermenting 1) can enable cells to adapt to changes in carbon metabolism by phosphorylating key rate-limiting enzymes involved in the maintenance of energy homeostasis and carbon metabolism within cells. Histone acetylation is an important epigenetic regulatory mechanism that influences physiological and biochemical processes. GCN5 is a representative histone acetylase involved in promoter chromatin remodeling and associated transcriptional activation. Here, the TvSNF1 and TvGCN5 genes were identified in Trichoderma viride Tv-1511, which exhibits promising activity with respect to its ability to produce cellulolytic enzymes for biological transformation. The SNF1-mediated activation of the histone acetyltransferase GCN5 was herein found to promote cellulase production in T. viride Tv-1511 via facilitating changes in histone acetylation. These results demonstrated that cellulolytic enzyme activity and the expression of genes encoding cellulases and transcriptional activators were clearly enhanced in T. viride Tv-1511 mutants in which TvSNF1 and TvGCN5 were overexpressed, with concomitant changes in histone H3 acetylation levels associated with these genes. GCN5 was also found to be directly recruited to promoter regions to alter histone acetylation, while SNF1 functioned upstream as a transcriptional activator that promotes GCN5 upregulation at the mRNA and protein levels in the context of cellulase induction in T. viride Tv-1511. These findings underscore the important role that this SNF1-GCN5 cascade plays in regulating cellulase production in T. viride Tv-1511 by promoting altered histone acetylation, offering a theoretical basis for the optimization of T. viride in the context of industrial cellulolytic enzyme production. KEY POINTS: • SNF1 kinase and GCN5 acetylase promoted cellulase production in Trichoderma by increasing the expression of genes encoding cellulases and transcriptional activators • SNF1 and GCN5 promoted cellulase production by driving H3ac modifications, and GCN5 directly band to the promoter regions to catalyze distinct H3ac modifications • SNF1 acts upstream of GCN5 as a transcriptional activator in the cellulase production of Trichoderma.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Multipurpose cellulases of Promicromonospora sp. VP111, with broad substrate specificity and tolerance properties.

Cellulolytic actinobacterium, Promicromonospora sp. VP111 concomitantly produced cellulases (CELs), xylanase and pectinase when grown on commercial cellulose and untreated agricultural lignocellulosic residues (wheat straw and sugarcane bagasse). Secreted CELs hydrolyzed (enhanced with Co2+ ion) multiple cellulosic substrates, including sodium carboxymethyl cellulose (Na-CMC), Whatman filter paper no. 1, microcrystalline cellulose (avicel), p-nitrophenyl-ß-D-glucopyranoside (pNPG), laminarin, and cellulose powder. The CELs showed stabilities in the presence of various chemicals, including glucose (0.2 M), detergents (1%, w/v or v/v), denaturants (1%, w/v or v/v), and sodium chloride (NaCl, 30%, w/v). The CELs were fractionated using ammonium sulfate precipitation and dialysis. Activities (%) of fractionated CELs were retained at 60°C for endoglucanase/carboxymethyl cellulase (CMCase) (88.38), filter paper cellulase (FPase) (77.55), and ß-glucosidase (90.52), which indicated of thermo-stability. Similarly, the activities (%) for CMCase (85.79), FPase (82.48), and ß-glucosidase (85.92) at pH 8.5 indicated of alkaline-stability. Kinetic factors, Km and Vmax for endoglucanase component of fractionated CELs were 0.014 g/l and 158.23 µM glucose/min/mL, respectively. Fractionated CELs yielded activation energies (kJ/mol) of 17.933, 6.294, and 4.207 for CMCase, FPase, and ß-glucosidase activities, respectively in linear thermostable Arrhenius plots. Thus, this study reports on the multipurpose CELs from an untreated agricultural residue utilizing Promicromonospora in relation to broad substrate specificity, halo-tolerance, alkaline-tolerance, detergent-tolerance, thermo-tolerance, organic solvent-tolerance, and end product-tolerance.

Potential of vermicomposting with mixtures of animal manure and vegetable leaves in the development of Eisenia foetida, microbial biomass, and enzymatic activity under semi-arid conditions.

Vermicomposting is the bio-oxidation and stabilization of organic matter involving relationships between the action of earthworms and microorganisms and the activation and dynamics of several enzyme activities. Semi-arid farmers to make (extra) money and organic production, produce their vermicompost using plant residues and animal manure, but there is no information about the final product generated. Thus, this study aimed to analyze the potential of vermicomposting with mixtures of animal manure and vegetable leaves in the development of Eisenia foetida, microbial biomass, and enzymatic activity in the semi-arid region, Brazil. The experimental design applied was randomized block in a 6 × 4 factorial scheme with four replicates, with six treatments (mixtures of cattle manure, goat manure, cashew leaves, and catanduva leaves) and evaluated at four-time intervals (30, 60, 90, and 120 days of vermicomposting). The treatments were placed in polyethylene pots in the same site, environmental conditions, and residues proportions as used by farmers. The characteristics analyzed were the number of earthworms (NE), total earthworm biomass (TEB) and earthworm multiplication index (MI), microbial biomass carbon (MBC), and activities of enzymes ß-glucosidase, dehydrogenase, alkaline and acid phosphatases. The cattle manure vermicomposted shows the highest average values observed for NE, MI, TEB, MBC, and enzymatic activity, regardless of the plant leaves mix. In general, the enzymes activities were found in the descending order of ß-glucosidase > alkaline phosphatase > dehydrogenase > acid phosphatase. The maturation dynamics of vermicompost were characterized by a decline in the microbial population and number and biomass of earthworms in the substrate and consequently a decrease in new enzyme synthesis and degradation of the remaining enzyme pool. Microbial biomass and enzymatic activity were indicators for changes in the quality of vermicompost.