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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Unveiling the role of long-range and short-range forces in the non-productive adsorption between lignin and cellulases at different temperatures.

Quantitatively understanding of interaction mechanism between lignin and cellulases is essential for the efficient improvement of lignocellulose enzymatic hydrolysis. However, the individual contribution of multiple forces between lignin and cellulases to the non-productive adsorption of enzymes still remains deeply ambiguous, especially in situations of near enzymatic hydrolysis temperatures. Herein, atomic force microscopy (AFM) and computational simulations were utilized to quantitatively analyze the intermolecular forces between lignin and enzyme at 25 °C and 40 °C. Our results unveiled that an increase in temperature obviously improved adsorption capacity and total intermolecular forces between lignin and cellulases. This positive relationship mainly comes from the increase in the decay length of hydrophobic forces for lignin-cellulases when temperature increases. Different from the hydrophobic interaction which provides long-range part of attractions, van der Waals forces dominate the intermolecular force only at approaches < 2 nm. On the other hand, electrostatic forces exhibited repulsive effects, and its intensity and distance were limited due to the low surface potential of cellulases. Short-range forces including hydrogen bonding (main) and π-π stacking (minor) stabilize the non-specific binding of enzymes to lignin, but increasing temperature reduces hydrogen bond number. Therefore, the relative contribution of long-range forces increased markedly at higher temperatures, which benefits protein capture and brings lignin and cellulase close together. Finally, the structure-activity relationships between lignin physicochemical properties and its inhibitory effect to enzymes indicated that hydrophobic interactions, hydrogen bonding, and steric effects drive the final adsorption capacity and glucose yields. This work provides quantitative and basic insights into the mechanism of lignin-cellulase interfacial interactions and guides design of saccharification enhancement approaches.

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.

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.

Alkalihalobacillus clausii PA21 transcriptome profiling and functional analysis revealed the metabolic pathway involved in glycoalkaloids degradation.

Glycoalkaloids (GAs), including α-solanine and α-chaconine, are secondary metabolites found in potato, which are toxic to higher animals. In a previous study, Alkalihalobacillus clausii PA21 showed the capacity to degrade GAs. Herein, the transcriptome response of PA21 to α-solanine or α-chaconine was evaluated. In total, 3170 and 2783 differential expressed genes (DEGs) were found in α-solanine- and α-chaconine-treated groups, respectively, with most DEGs upregulated. Moreover, GAs activated transmembrane transport, carbohydrate metabolism, transcription, quorum sensing, and bacterial chemotaxis in PA21 to withstand GA-induced stress and promote GAs degradation. Furthermore, qRT-PCR analysis confirmed the upregulation of degrading enzymes and components involved in GA degradation in PA21. In addition, the GAs-degrading enzymes were heterologous expressed, purified, and incubated with GAs to analyze the degradation products. The results showed that α-solanine was degraded to ß1-solanine, ß2-solanine, γ-solanine, and solanidine by ß-glucosidase, α-rhamnosidase, and ß-galactosidase. Meanwhile, α-chaconine was degraded to ß1-chaconine, ß2-chaconine, γ-chaconine, and solanidine by ß-glucosidase and α-rhamnosidase. Overall, the molecular mechanism underlying GAs degradation by PA21 was revealed by RNAseq combined with protein expression and function studies, thus providing the basis for the development of engineered bacteria that can efficiently degrade GAs to promote their application in the control of GAs in potatoes.

Millipede gut-derived microbes as a potential source of cellulolytic enzymes.

Lignocellulose biomass has recently been considered a cost-effective and renewable energy source within circular economy management. Cellulases are important key enzymes for simple, fast, and clean biomass decomposition. The intestinal tract of millipedes is the environment which can provide promising microbial strains with cellulolytic potential. In the present study, we used the tropical millipede Telodeinopus aoutii as an experimental organism. Within a feeding test in which millipedes were fed with oak and maple leaf litter, we focused on isolating culturable cellulolytic microbiota from the millipede gut. Several growth media selecting for actinobacteria, bacteria, and fungi have been used to cultivate microbial strains with cellulolytic activities. Our results showed that oak-fed millipedes provided a higher number of culturable bacteria and a more diversified microbial community than maple-fed ones. The screening for cellulolytic activity using Congo red revealed that about 30% of bacterial and fungal phylotypes isolated from the gut content of T. aoutii, produced active cellulases in vitro. Actinobacteria Streptomyces and Kitasatospora were the most active cellulolytic genera on Congo red test. In contrast, fungi Aspergillus, Penicillium, Cheatomium, Clonostachys, and Trichoderma showed the highest protein-specific cellulase activity quantified by 4-Methylumbelliferyl ß-D-cellobioside (4-MUC). Our findings provide a basis for future research on the enzyme activities of microbes isolated from the digestive tracts of invertebrates and their biocatalytic role in biomass degradation.

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.