Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology.

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.

Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production.

BACKGROUND: Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. METHODS AND RESULT: The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936 bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. CONCLUSION: A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later.

The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum.

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at "CTGGGG". In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

UCST-Type Soluble Immobilized Cellulase: A New Strategy for the Efficient Degradation and Improved Recycling Performance of Wastepaper Cellulose.

This paper reports an innovative study that aims to address key issues in the efficient recycling of wastepaper cellulose. The research team utilized the temperature-responsive upper critical solution temperature (UCST) polymer P(NAGA-b-DMA) in combination with the LytA label's affinity for choline analogs. This innovative approach enabled them to successfully develop a novel soluble immobilized enzyme, P(NAGA-b-DMA)-cellulase. This new enzyme has proven highly effective, significantly enhancing the degradation of wastepaper cellulose while demonstrating exceptional stability. Compared with the traditional insoluble immobilized cellulase, the enzyme showed a significant improvement in the pH, temperature stability, recycling ability, and storage stability. A kinetic parameter calculation showed that the enzymatic effectiveness of the soluble immobilized enzyme was much better than that of the traditional insoluble immobilized cellulase. After the immobilization reaction, the Michaelis constant of the immobilized enzyme was only increased by 11.5%. In the actual wastepaper degradation experiment, the immobilized enzyme was effectively used, and it was found that the degradation efficiency of wastepaper cellulose reached 80% of that observed in laboratory conditions. This novel, thermosensitive soluble immobilized cellulase can efficiently catalyze the conversion of wastepaper cellulose into glucose under suitable conditions, so as to further ferment into environmentally friendly biofuel ethanol, which provides a solution to solve the shortage of raw materials and environmental protection problems in the paper products industry.

Study on the degradation conditions of corn stalks by Asian corn borer digestive enzymes combined with white-rot fungus.

Since the environmentally friendly reuse of corn stalks attracts more and more attention, it is an efficient and feasible way to reuse corn stalks as forage. However, whether the cellulose, lignin, and hemicellulose within corn stalks can be effectively decomposed becomes a key to reusing corn stalks as forage. Orthogonal test was designed by five different degradation temperatures (22°C, 24°C, 26°C, 28°C, 30°C), five different pH values (4, 5, 6, 8, 10), and five different degradation time durations (5, 15, 25, 30, and 35 days) to examine 25 kinds of different degradation conditions. It was found that the decomposition effect of hemicellulose, cellulose, and lignin, of group 25 (26°C, pH = 5, 25 days) was stronger compared with other groups, with the contents calculated as 8.22%, 31.55%, and 22.55% individually (p < 0.01, p < 0.05). Group 19 (22°C, pH = 4, 5 days) revealed the worst degradation effect of cellulose, lignin, and hemicellulose compared to other groups, with contents calculated as 15.48%, 38.85%, and 29.57%, individually (p < 0.01, p < 0.05). The research data deliver a basis for ideal degradation conditions for corn stalks degradation in combination with the digestive enzymes of P. chrysosporium and O. furnacalis larva. Aiming to explore a highly efficient and environmentally friendly corn stalk degradation method.

Investigation of anti-adherence and antimicrobial properties of prodigiosin-functionalized bacterial cellulose membrane for biomedical applications.

In this study, novel biomaterial that consisted entirely of bacterial products was developed with the approach of designing cost effective material for biomedical applications. With this aim, bacterial cellulose membranes (BCMs) which synthesized by Komagataeibacter intermedius were produced. Moreover, to impart antimicrobial properties to enhance the capacity of BCMs for biomedical usage, prodigiosin (PG) pigment of Serratia marcescens which presents wide range of antimicrobial activities was loaded to BCMs. Firstly, high yield of PG production was achieved, and then crude pigment was purified with silica gel column. The purified PG was characterized with thin layer chromatography and UV-visible spectrometry. The antimicrobial effect of the produced pigment on Gram-positive and negative bacteria and a yeast was investigated. The success of modification in PG-modified BCMs has been demonstrated by FTIR and SEM. Moreover, antimicrobial and antiadhesive ability of novel PG-BCMs were examined with disc diffusion and plate counting methods. As a result, it was established that PG-BCMs were able to inhibit the growth of all tested microorganisms. Furthermore, excellent antiadhesive effect was observed for the tested microorganisms with the inhibition rates of 82.05-96.25 %. Finally, cytotoxicity test with L929 cell line demonstrated that PG-BCM is biocompatible at a level that can be applied in in vivo studies.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Complete genome sequence and transcriptome response to vitamin C supplementation of Novacetimonas hansenii SI1 - producer of highly-stretchable cellulose.

Novacetimonas hansenii SI1, previously known as Komagataeibacter hansenii, produces bacterial nanocellulose (BNC) with unique ability to stretch. The addition of vitamin C in the culture medium increases the porosity of the membranes and their stretchability making them highly moldable. To better understand the genetic background of this strain, we obtained its complete genome sequence using a hybrid sequencing and assembly strategy. We described the functional regions in the genome which are important for the synthesis of BNC and acetan-like II polymer. We next investigated the effect of 1% vitamin C supplementation on the global gene expression profile using RNA sequencing. Our transcriptomic readouts imply that vitamin C functions mainly as a reducing agent. We found that the changes in cellular redox status are balanced by strong repression of the sulfur assimilation pathway. Moreover, in the reduced conditions, glucose oxidation is decreased and alternative pathways for energy generation, such as acetate accumulation, are activated. The presence of vitamin C negatively influences acetan-like II polymer biosynthesis, which may explain the lowered yield and changed mechanical properties of BNC. The results of this study enrich the functional characteristics of the genomes of the efficient producers of the N. hansenii species. Improved understanding of the adaptation to the presence of vitamin C at the molecular level has important guiding significance for influencing the biosynthesis of BNC and its morphology.

Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins.

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.

The orchestration of sustained drug delivery by bacterial cellulose/gelatin nanocomposites reinforced with carboxylic carbon nanotubes.

Recently, modifying bacterial cellulose (BC) by compositing it with other nano-biomaterials has become inevitable to achieve its desired properties in drug delivery. To address this, our study endeavors to utilize an in-situ fabrication method for the creation of a multifunctional BC/gelatin (BC/Gel) platform reinforced with carboxylic multi-walled carbon nanotubes (cMWCNTs) as a sustainable delivery model of biomolecules. Incipiently, cMWCNTs were loaded with human serum albumin (HSA) as a drug model, with an optimized nanoparticle-to-protein ratio of 1:5 and loading efficiency of 90.0 ± 1.0 % before incorporation into BC/Gel hydrogels. By comparison, nanocomposition improved the surface area and overall porosity of BC/Gel up to 58.0 ± 1.3 m2/g and 85.5 ± 1.1 %, respectively. Likewise, significant wettability of 44.0 ± 0.1° and dramatic biodegradation rate of 36.9 ± 1.2 % were other exceptionally gained attributes. Meanwhile, with a Zero-order kinetic mechanism, CNT-HSA integration facilitated the controlled release of 56.0 ± 0.9 % HSA over 7 days. Drug-loaded nanocomposites showcased >70 % viability during in vitro cellular trials using Human Foreskin Fibroblasts (HFF). Overall, BC/Gel/CNT-HSA nanocomposite exhibited favorable cell behavior, devoid of cytotoxic manifestations. Consequently, this BC-based nanocomposite scaffold implicates the premiere capability in the sustained delivery of an extended range of protein biomolecules, offering a promising therapeutic avenue for bolstering tissue regeneration.

Engineering Yarrowia lipolytica as a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose.

De novo biosynthesis of high-value added food additive p-coumaric acid (p-CA) direct from cellulose/hemicellulose is a more sustainable route compared to the chemical route, considering the abundant cellulose/hemicellulose resources. In this study, a novel factory was constructed for the production of p-CA in Yarrowia lipolytica using cellulose/hemicellulose as the sole carbon source. Based on multicopy integration of the TAL gene and reprogramming the shikimic acid pathway, the engineered strain produced 1035.5 ± 67.8 mg/L p-CA using glucose as a carbon source. The strains with overexpression of cellulases and hemicellulases produced 84.3 ± 2.4 and 65.3 ± 4.6 mg/L p-CA, using cellulose (carboxymethyl-cellulose) or hemicellulose (xylan from bagasse) as the carbon source, respectively. This research demonstrated the feasibility of conversion of cost-effective cellulose/hemicellulose into a value-added product and provided a sustainable cellulolytic cell factory for the utilization of cellulose/hemicellulose.

Full-fat corn germ improves the performance and milk fat yield of Girolando cows fed sugarcane bagasse and cactus cladodes as forage sources.

We investigated the effects of replacing ground corn with full-fat corn germ (FFCG) on milk production, milk composition, and nutrient use in cows fed sugarcane bagasse and cactus cladodes. Ten multiparous Girolando cows (average body weight 500 ± 66 kg, 90 ± 15 days in milk) were distributed in a replicated 5 × 5 Latin Square and assigned to five dietary treatments containing 0%, 25%, 50%, 75%, or 100% of full-fat corn germ in substitution to ground corn. Full-fat corn germ increased fat-corrected milk yield by 2.2 kg/day and the synthesis of fat, lactose, and total solids in milk by 94.4, 60.0, and 201.10 g/day, respectively (p < 0.05). Cows fed corn germ quadratically increased (p < 0.05) dry matter intake by 1.01 kg/day, with the intake of crude protein and total digestible nutrients following the same pattern. Conversely, the substitution of corn for full-fat corn germ linearly reduced (p < 0.05) the total non-fiber carbohydrate intake from 5.79 to 4.40 kg/d. Except for ether extract and non-fiber carbohydrates, full-fat corn germ did not alter (p > 0.05) nutrient digestibility. Cows fed corn germ excreted less (p < 0.05) urea-N in milk and urine N. These results demonstrate that full-fat corn germ can partially replace ground corn to enhance the milk production efficiency of crossbred cows fed cactus cladodes and sugarcane bagasse. Furthermore, including sugarcane bagasse in FFCG-supplemented diets prevents milk fat depression in cows fed cactus cladodes.

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Cryptic diversity of cellulose-degrading gut bacteria in industrialized humans.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Effects of heterologous expression and N-glycosylation on the hyperthermostable endoglucanase of Pyrococcus furiosus.

Hyperthermostable endoglucanases of glycoside hydrolase family 12 from the archaeon Pyrococcus furiosus (EGPf) catalyze the hydrolysis of ß-1,4-glucosidic linkages in cellulose and ß-glucan structures that contain ß-1,3- and ß-1,4-mixed linkages. In this study, EGPf was heterologously expressed with Aspergillus niger and the recombinant enzyme was characterized. The successful expression of EGPf resulted as N-glycosylated protein in its secretion into the culture medium. The glycosylation of the recombinant EGPf positively impacted the kinetic characterization of EGPf, thereby enhancing its catalytic efficiency. Moreover, glycosylation significantly boosted the thermostability of EGPf, allowing it to retain over 80% of its activity even after exposure to 100 °C for 5 h, with the optimal temperature being above 120 °C. Glycosylation did not affect the pH stability or salt tolerance of EGPf, although the glycosylated compound exhibited a high tolerance to ionic liquids. EGPf displayed the highest specific activity in the presence of 20% (v/v) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), reaching approximately 2.4 times greater activity than that in the absence of [Bmim]Cl. The specific activity was comparable to that without the ionic liquid even in the presence of 40% (v/v) [Bmim]Cl. Glycosylated EGPf has potential as an enzyme for saccharifying cellulose under high-temperature conditions or with ionic liquid treatment due to its exceptional thermostability and ionic liquid tolerance. These results underscore the potential of N-glycosylation as an effective strategy to further enhance both the thermostability of highly thermostable archaeal enzymes and the hydrolysis of barley cellulose in the presence of [Bmim]Cl.

Microbial fermentation and black soldier fly feeding to enhance maize straw degradation.

This study used an innovative synergistic microbial and insect approach to treat maize straw and kitchen waste substrates, including cyclic microbial fermentation and feeding of black soldier fly larvae (BSFL) using the fermented substrate. Increasing cycle numbers led to significantly increased cellulose, hemicellulose, and lignin degradation rates (DR) in the maize straw, which increased by 68.28%, 81.43% and 99.95%, respectively, compared to those in the blank group without frass addition. Moreover, according to the experimental results, it was revealed that the structure of lignocellulose, the composition and structure of the bacterial community in the BSFL gut and frass changed significantly after the addition of the previous cycle of frass treatment. Moreover, the differences in amplicon sequence variants (ASVs) between the gut and frass further increased. The relative abundances of Enterococcus and Actinobacteria in the gut and Gammaproteobacteria_unclassified and Dysgonomonas in the frass increased significantly, which may play a more positive role in lignocellulose degradation. In conclusion, this study showed that frass fermentation + BSFL feeding to degrade straw is a promising method and that frass fermentation is beneficial for the whole cycle. Furthermore, these findings underscore the beneficial impact of frass fermentation on the entire cycle.