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Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high‐coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S. microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty‐six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S. microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well‐known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S. microspora may have potential for use in the biofuel, textile, and detergent industries. The goal of this study was to obtain a high‐coverage draft of the genome of the rare alkaline‐halophilic Stachybotrys microspora and to annotate the genome sequence. Using two bioinformatics tools, fungicompanion and OmicsBox, we identified several cellulase genes, suggesting this species may have potential for use in several industries.

Cellulase is essential for cellulose hydrolysis and is used across industries such as food, feed, textiles, biofuel, and biomass pretreatment. However, its utility is restricted by high temperatures and salt concentrations. This study identified a novel cellulase gene (named c5-cel4 ) from Ebinur Salt Lake in Xinjiang, China using metagenomic technology. Its amino acid sequence has a 90.97% similarity to the GH5 family cellulase of Microbulbifer litoralis (WP_250463697.1). The gene was expressed in Escherichia coli , and the recombinant protein, C5-CEL4, was purified via Ni-NTA affinity chromatography. C5-CEL4, secreted extracellularly (0.886 U/mL), revealed a protein size 14 KDa smaller than predicted, with mass spectrometry indicating a truncated C-terminal of 135 amino acid residues. Optimal activity was observed at 50 °C and pH 7.0, maintaining over 80% activity at 40–65 °C and 45–50 °C for 2 h. The enzyme’s half-life was 60 min at 55–60 °C, retaining over 90% activity after 24 h in pH 5.0–12.0 buffers. C5-CEL4 showed activity against CMC-Na, bagasse xylan, and beech xylan, with Kcat values of 98.20 s − 1 and 12.32 s − 1 for CMC-Na and bagasse xylan, respectively. It also hydrolyzed wheat bran and maize stalks into reducing sugars. Remarkably, C5-CEL4 exhibited high salt tolerance, maintaining 100% activity in 0.5 M-5.0 M NaCl and after 9 months in 5.0 M NaCl. It retained over 90% activity in ionic liquids (BMIM-Ac, EMIM-Cl, BMIM-BF4) at 50% concentration and showed resistance to heavy metal ions (Co 2+ , Cu 2+ , Ag + , Mn 2+ , Pb 2+ , and Ni 2+ ) and inhibitors (PMSF, DTT, Tween80, and SDS). In conclusion, C5-CEL4 is a robust cellulase with heat, alkali, salt, ionic liquid, and inhibitor resistance, alongside cellulase and xylanase activity, presenting significant potential for feed, food, and bioenergy applications.

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular β-glucosidase enzymes and one intracellular β-glucosidase lacks β-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple β-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

In this paper, we reported a simple and efficient protocol for preparation of Cu super(2+)-modified magnetic Fe sub(3)O sub(4)iO sub(2 ) core/shell microspheres for immobilization of cellulase. The uniform magnetic Fe sub(3)O sub(4)iO sub(2 ) core/shell microspheres with a thin shell of 20 nm were synthesized through a solvothermal method followed by a sol-gel process. An amino-terminated silane coupling agent of (3-aminopropyl)triethoxysilane (APTS) was then grafted on them for capturing Cu super(2+) ions. The reaction process is very simple, efficient, and economical. Noticeably, the content of Cu super(2+) ions on the magnetic core/shell microspheres can reach 4.6 Wt%, endowing them possess as high immobilization capacity as 225.5 mg/g for cellulase. And the immobilized cellulase can be retained over 90 % on the magnetic microspheres after six cycles. Meanwhile, the magnetic microspheres decorated with Cu super(2+) ions show a superparamagnetic character with a high magnetic saturation of 58.5 emu/g at room temperature, suggesting conveniently and rapidly recycle the enzyme from solution. This facile, recyclable, high immobilization capacity and activity strategy may find potential applications in enzyme catalytic reactions with low cost.

Lignin is central to overcoming recalcitrance in the enzyme hydrolysis of lignocellulose. While the term implies a physical barrier in the cell wall structure, there are also important biochemical components that direct interactions between lignin and the hydrolytic enzymes that attack cellulose in plant cell walls. Progress toward a deeper understanding of the lignin synthesis pathway – and the consistency between a range of observations over the past 40 years in the very extensive literature on cellulose hydrolysis – is resulting in advances in reducing a major impediment to cellulose conversion: the cost of enzymes. This review addresses lignin and its role in the hydrolysis of hardwood and other lignocellulosic residues. Lignin and lignin-derived phenolic compounds inhibit lignocellulolytic enzymes. While lignin nonspecifically adsorbs enzymes, phenolic compounds inhibit and/or deactivate them. The effect will vary depending on type of phenolic compounds, their concentration, and possible synergistic effects. The effect will also depend on the type of enzyme and microorganism from which they were produced. Enzymes from Trichoderma reesei are more susceptible to the inhibitory and/or deactivating effects than those from Aspergillus niger. Noncatalytic proteins, such as bovine serum albumin or soy-derived proteins, minimize the nonspecific adsorption of the hydrolytic enzymes on lignin. Understanding these mechanisms of enzyme inhibition or deactivation and approaches to mitigate them potentially favor continued reduction of cellulose conversion-associated costs.

Despite the lack of implementation of consolidated bioprocessing (CBP) at an industrial scale, this bioconversion strategy still holds significant potential as an economically viable solution for converting lignocellulosic biomass (LCB) into biofuels and green chemicals, provided an appropriate organism can be isolated or engineered. The use of Saccharomyces cerevisiae for this purpose requires, among other things, the development of a cellulase expression system within the yeast. Over the past three decades, numerous studies have reported the expression of cellulase-encoding genes, both individually and in combination, in S. cerevisiae . Various strategies have emerged to produce a core set of cellulases, with differing degrees of success. While one-step conversion of cellulosic substrates to ethanol has been reported, the resulting titers and productivities fall well below industrial requirements. In this review, we examine the strategies employed for cellulase expression in yeast, highlighting the successes in developing basic cellulolytic CBP-enabled yeasts. We also summarize recent advancements in rational strain design and engineering, exploring how these approaches can be further enhanced through modern synthetic biology tools to optimize CBP-enabled yeast strains for potential industrial applications. Key points • S. cerevisiae’s lack of cellulolytic ability warrants its engineering for industry. • Advancements in the expression of core sets of cellulases have been reported. • Rational engineering is needed to enhance cellulase secretion and strain robustness. • Insights gained from omics strategies will direct the future development of CBP strains. Graphical Abstract

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

The present study focuses on the isolation and characterization of a bacterium from Lake Van adapted to an alkaline pH environment with significant cellulase production potential. The identified isolate, Bacillus pumilus VLC7 (GenBank Acc No: OR415888.1), exhibited the highest cellulase activity among other alkaliphilic isolates. The purification employing protein precipitation by ammonium sulfate, ultrafiltration, and ion exchange chromatography resulted in 20-fold purification with a specific activity of 16 U/mg protein. Cellulase had a molecular weight of 76 kDa, 3.13 mM K m and 0.160 U/mg V max values. The enzyme displayed maximum activity at pH 9.0 and 40 °C and retained at least 80% of its activity at temperatures of 25–60 °C for 90 min and pH 4–12 for one hour. Stability tests also revealed the enzyme’s resilience to various reagents, metal ions, organic solvents, and detergents. Furthermore, the biotechnological applications of cellulase were explored, demonstrating its effectiveness in fabric biopolishing (removing pilling), as well as in the removal of fabric dyes. The research findings underscore the potential of B. pumilus VLC7 as a valuable source for eco-friendly industrial biotechnology applications. Graphical Abstract

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars.

Thermophilic cellulases can play a crucial part in the efficient breakdown of cellulose—a major component of lignocellulosic plant biomass, however, their commercial production needs simple and robust biomanufacturing biosystems. In this study, two cellulases (β-glucosidase and endoglucanase) were heterologously expressed in Escherichia coli under a chloroplast-derived constitutive promoter and expression-enhancing terminator. The genes encoding the cellulases were sourced from a thermophilic bacterium Thermotoga maritima to exploit their industrially needed thermotolerance potential. The codon-optimized gene sequences were synthesized and placed under a tobacco chloroplast 16S rRNA promoter (Prrn), along with the 5′ UTR (untranslated region) from gene 10 of phage T7 (T7g10). A six-residue long histidine tag (His 6 -tag) was attached to the N-terminus for protein detection. A high-level of expression of β-glucosidase and endoglucanase in E. coli was recorded from the chloroplast promoter and terminator. Furthermore, the activity assays confirmed that the recombinant enzymes maintained their activity at elevated temperatures. Thermostability analysis showed that recombinant enzymes retained their thermotolerance even after being expressed in a non-native host. Where, β-glucosidase and endoglucanase showed their optimum activities at 90 °C and 100 °C, respectively. Examination of the 3D structures of T. maritima cellulases revealed differential ionic interactions contributing to this high degree of thermotolerance. The study highlights the feasibility of producing thermostable versions of recombinant enzymes in E. coli at high levels. Our finding underscores the potential of this approach to meet industrial demands for efficient enzyme production employing E. coli as a robust biomanufacturing platform.