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Background Plant endoglucanases of glycosyl hydrolase family 9 (GH9), which cleave 1,4-β-glucosidic bonds in glycan chains, have been shown to play pivotal roles in cellulose degradation, as well as in the relaxation or construction of cell walls during diverse plant growth and developmental processes. However, their specific functions in plant sexual reproduction remain unclear. Results In this study, we systematically analyzed expression profiles of the GH9 gene family using in vivo observations of GFP-translational fusion proteins. Our results revealed that numerous GH9 genes were expressed across both vegetative and reproductive tissues: 5 in stomata, 12 in roots, 11 in mature pollen and 15 in mature ovules or seeds. Further analysis of reproductive tissues uncovered distinct expression specificities: GH9A4 as pollen-specific, GH9B6 as sperm cell-specific, GH9B12 as central cell- and endosperm cell-specific, GH9B11 as enriched in pollen and chalazal endosperm, and GH9B8 as vascular tissue-preferential. Additionally, GFP signals of up to 7 GH9 members were detected in the filiform apparatus, while 12 GH9 members showed signals in integuments and seed coats—hinting at diverse GH9 functions in reproduction. Furthermore, we conducted gene editing and phenotypic analysis on two subsets of GH9 genes highly expressed in pollen. While in vivo pollen germination and growth were unaffected, in vitro germination rates decreased significantly when GH9B5 , GH9B7 , and GH9A4 functionally deficient. Conclusions Taken together, this study highlights the potential roles of the GH9 family in pollen germination and establishes a foundation for future functional analyses aimed at elucidating the putative roles of the GH9 family in plant reproduction.

Background Genetic modification of plant cell walls has been implemented to reduce lignocellulosic recalcitrance for biofuel production. Plant glycoside hydrolase family 9 (GH9) comprises endo-β-1,4-glucanase in plants. Few studies have examined the roles of GH9 in cell wall modification. In this study, we independently overexpressed two genes from GH9B subclasses (OsGH9B1 and OsGH9B3) and examined cell wall features and biomass saccharification in transgenic rice plants. Results Compared with the wild type (WT, Nipponbare), the OsGH9B1 and OsGH9B3 transgenic rice plants, respectively, contained much higher OsGH9B1 and OsGH9B3 protein levels and both proteins were observed in situ with nonspecific distribution in the plant cells. The transgenic lines exhibited significantly increased cellulase activity in vitro than the WT. The OsGH9B1 and OsGH9B3 transgenic plants showed a slight alteration in three wall polymer compositions (cellulose, hemicelluloses, and lignin), in their stem mechanical strength and biomass yield, but were significantly decreased in the cellulose degree of polymerization (DP) and lignocellulose crystalline index (CrI) by 21–22%. Notably, the crude cellulose substrates of the transgenic lines were more efficiently digested by cellobiohydrolase (CBHI) than those of the WT, indicating the significantly increased amounts of reducing ends of β-1,4-glucans in cellulose microfibrils. Finally, the engineered lines generated high sugar yields after mild alkali pretreatments and subsequent enzymatic hydrolysis, resulting in the high bioethanol yields obtained at 22.5% of dry matter. Conclusions Overproduction of OsGH9B1/B3 enzymes should have specific activity in the postmodification of cellulose microfibrils. The increased reducing ends of β-1,4-glucan chains for reduced cellulose DP and CrI positively affected biomass enzymatic saccharification. Our results demonstrate a potential strategy for genetic modification of cellulose microfibrils in bioenergy crops.

Background Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. Results The primary aim of this study was to discover a novel alkali-thermostable endo-β-1,4-glucanase from the sheep rumen metagenome. At first, the multi-step in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the computationally selected candidates, PersiCel4 was found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes’ kinetic parameters, including V max , K m , and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85 °C and was able to retain more than 70% of its activity after 150 h of storage at 85 °C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl and several metal ions contains Mg 2+ , Mn 2+ , Cu 2+ , Fe 2+ and Ca 2+ . Our results showed that treatment with MnCl 2 could enhance the enzyme’s activity by 78%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 h in the harsh condition (T = 85 °C, pH = 8.5). Conclusion Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.

Termites live in colonies, and their members belong to different castes that each have their specific role within the termite society. In well-established colonies of higher termites, the only food the founding female, the queen, receives is saliva from workers; such queens can live for many years and produce up to 10,000 eggs per day. In higher termites, worker saliva must thus constitute a complete diet and therein resembles royal jelly produced by the hypopharyngeal glands of honeybee workers that serves as food for their queens; indeed, it might as well be called termite royal jelly. However, whereas the composition of honeybee royal jelly is well established, that of worker termite saliva in higher termites remains largely unknown. In lower termites, cellulose-digesting enzymes constitute the major proteins in worker saliva, but these enzymes are absent in higher termites. Others identified a partial protein sequence of the major saliva protein of a higher termite and identified it as a homolog of a cockroach allergen. Publicly available genome and transcriptome sequences from termites make it possible to study this protein in more detail. The gene coding the termite ortholog was duplicated, and the new paralog was preferentially expressed in the salivary gland. The amino acid sequence of the original allergen lacks the essential amino acids methionine, cysteine and tryptophan, but the salivary paralog incorporated these amino acids, thus allowing it to become more nutritionally balanced. The gene is found in both lower and higher termites, but it is in the latter that the salivary paralog gene got reamplified, facilitating an even higher expression of the allergen. This protein is not expressed in soldiers, and, like the major royal jelly proteins in honeybees, it is expressed in young but not old workers.

Cellulase plays an irreplaceable role in biomanufacturing using plant biomass as feedstock. However, improving cellulase production by fungi through manipulation of upstream open reading frames (uORFs) in the 5′-untranslated regions (5′-UTR) of cellulase genes has been less frequently explored. This study aimed to screen uORFs in the 5′-UTR of cellulase genes in Penicillium oxalicum, identify functional uORFs in the 5′-UTR of the eg1 gene which encodes a key endo-β-1,4-glucanase (EG) in P. oxalicum, and enhance fungal cellulase production through uORF modifications. Among the 25 cellulase genes examined in P. oxalicum strain HP7-1, 23 contained uORFs in their 5′-UTR. Seven uORFs were annotated in the 5′-UTR of the eg1 gene. A uORF-green fluorescent protein (GFP) reporter system demonstrated that uORF1 and uORF3 inhibited, while uORF7 enhances, GFP abundance. Overexpression of eg1 containing uORF1 or uORF3 variants where the start codon of the uORF was mutated to AAG in P. oxalicum led to a significant 91.7 % and 62.1 % average increase in carboxymethyl cellulase production after 4 days of induction compared to the start strain ΔPoxKu70. Real-time quantitative reverse transcription-polymerase chain reaction, mRNA stability determination, and in vitro translation experiments collectively revealed that these three uORFs influence the mRNA stability of the downstream mORF, but not translation efficiency. These findings highlight the critical role of uORFs in regulating gene expression during fungal enzyme biosynthesis and offer a valuable alternative strategy for improving enzyme production. [Display omitted]

Genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance for enhanced biomass enzymatic saccharification and biofuel production in bioenergy crops. Although endo-β-1,4-glucanase (EG II) secreted by fungi has been broadly applied for enzymatic hydrolysis of cellulose, it remains to explore its role in cellulose modification when the EG II gene is overexpressed in plant. In this study, we selected transgenic rice plants that overproduced Trichoderma reesei EG II enzyme specifically deposited into plant cell walls, and then examined much higher enzymatic activities by fourfold to fivefold in transgenic young seedlings than those of wild type in vitro. Notably, despite slightly altered cell wall compositions and polymer interlinkages relative to the wild type, the transgenic mature rice straw exhibited significantly reduced cellulose DP and CrI values and hemicellulosic Xyl/Ara ratio, leading to much increased biomass porosity. These should play integrated impact for enhanced biomass enzymatic saccharification and bioethanol production even under mild alkali pretreatment. Therefore, the results suggested that the EG II deposition should have enzymatic activity specific for minor-modification of cellulose microfibrils in transgenic rice plants. It has also provided a potential strategy for mild cell wall modification and optimal biomass process in rice and other bioenergy crops.

The food enzyme cellulase (4‐(1,3; 1,4)‐β‐D‐glucan 4‐glucanohydrolase; EC 3.2.1.4) is produced with the genetically modified Trichoderma reesei strain AR‐715 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in seven food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in four processes, dietary exposure was calculated for the remaining three food manufacturing processes. It was estimated to be up to 0.070 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated‐dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 14,286. A search for the homology of the amino acid sequence of the cellulase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-β-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-β-1,4-glucanase gene (CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6–9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley β-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G4) and cellppentaose (G5) to cellobiose (G2) and cellotriose (G3). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications.

The gene (1608-bp) encoding a GH6 endo-β-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0–10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley β-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including β-1,3-D-glucan, β-1,4-D-galactomannan, and β-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley β-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30–50°C at a pH of 6–7. The thermostability was retained and the pH was stable in the range of 30–50°C at a pH of 5–7.