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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.

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

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.

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.

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.

This study reports the isolation and characterization of Bacillus subtilis K35-1, a novel cellulolytic strain with exceptional forage degradation capabilities. From eight B. subtilis isolates obtained from yak rumen fluid through Congo red screening (hydrolysis capacity = 2.61 ± 0.23), K35-1 demonstrated superior enzymatic performance, achieving peak cellulase (77.26 U/mL) and hemicellulase (222.85 nmol/min/mL) activities at 36 h of fermentation. The whole genome sequencing revealed a 4.06 Mb circular chromosome (GC content 43.83%) encoding 3,980 protein-coding sequences. Comprehensive CAZy annotation identified 703 carbohydrate-active enzymes, including: 87 cellulases spanning 7 GH families (GH5, GH6, GH9, GH12, GH44, GH45, GH48) and 34 hemicellulases from 4 GH families (GH10, GH11, GH26, GH30). Comparative genomic analysis showed K35-1 possesses 40% more glycoside hydrolases than reference strains (Srivastava et al., Mol Genet Genomics 298:361–74, 2023), explaining its enhanced degradation efficiency (53.2% cellulose reduction vs. 7.3% in conventional treatments). Functional annotation revealed: 275 carbohydrate metabolism genes (KEGG), 228 cell wall/membrane biogenesis genes (COG) and 53.91% reduced-virulence mutations (PHI database). The strain’s robust enzymatic profile, coupled with minimal antibiotic resistance (11 genes, including ermB ), positions K35-1 as both an efficient forage degrader and safe probiotic candidate. These findings provide a genomic foundation for developing novel feed additives to improve livestock nutrition.

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.

Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called \"mycodiesel,\" while growing on lignocellulosic plant and agricultural residues. The fact that endophytes are plant symbionts suggests that some may be able to produce lignocellulolytic enzymes, making them capable of both deconstructing lignocellulose and converting it into mycodiesel, two properties that indicate that these strains may be useful consolidated bioprocessing (CBP) hosts for the biofuel production. In this study, four endophytes Hypoxylon sp. CI4A, Hypoxylon sp. EC38, Hypoxylon sp. CO27, and Daldinia eschscholzii EC12 were selected and evaluated for their CBP potential. Analysis of their genomes indicates that these endophytes have a rich reservoir of biomass-deconstructing carbohydrate-active enzymes (CAZys), which includes enzymes active on both polysaccharides and lignin, as well as terpene synthases (TPSs), enzymes that may produce fuel-like molecules, suggesting that they do indeed have CBP potential. GC-MS analyses of their VOCs when grown on four representative lignocellulosic feedstocks revealed that these endophytes produce a wide spectrum of hydrocarbons, the majority of which are monoterpenes and sesquiterpenes, including some known biofuel candidates. Analysis of their cellulase activity when grown under the same conditions revealed that these endophytes actively produce endoglucanases, exoglucanases, and β-glucosidases. The richness of CAZymes as well as terpene synthases identified in these four endophytic fungi suggests that they are great candidates to pursue for development into platform CBP organisms.

Background Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. Results Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. Conclusions The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67–71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei.