Genome-wide investigation of glycoside hydrolase 9 (GH9) gene family unveils implications in orchestrating the mastication trait of Citrus sinensis fruits.

Mastication trait of citrus significantly influences the fruit's overall quality and consumer preference. The accumulation of cellulose in fruits significantly impacts the mastication trait of citrus fruits, and the glycoside hydrolase 9 (GH9) family plays a crucial role in cellulose metabolism. In this study, we successfully identified 32 GH9 genes from the Citrus sinensis genome and subsequently conducted detailed bioinformatics analyses of the GH9 family. Additionally, we profiled the spatiotemporal expression patterns of CsGH9 genes across four distinct fruit tissue types and six crucial developmental stages of citrus fruits, leveraging transcriptome data. Parallel to this, we undertook a comparative analysis of transcriptome profiles and cellulose content among diverse fruit tissues spanning six developmental stages. Furthermore, to identify the pivotal genes involved in cellulose metabolism within the GH9 family during fruit maturity, we employed correlation analysis between cellulose content and gene expression in varying tissues across diverse citrus varieties. This analysis highlighted key genes such as CsGH9A2/6 and CsGH9B12/13/14/22. Collectively, this study provides an in-depth analysis of the GH9 gene family in citrus and offers novel molecular insights into the underlying mechanisms governing the mastication trait formation in citrus fruits.

A processive GH9 family endoglucanase of Bacillus licheniformis and the role of its carbohydrate-binding domain.

One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A, encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli. The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as ß-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. KEY POINTS: • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase.

Genome Wide Identification and Characterization of Wheat GH9 Genes Reveals Their Roles in Pollen Development and Anther Dehiscence.

Glycoside hydrolase family 9 (GH9) is a key member of the hydrolase family in the process of cellulose synthesis and hydrolysis, playing important roles in plant growth and development. In this study, we investigated the phenotypic characteristics and gene expression involved in pollen fertility conversion and anther dehiscence from a genomewide level. In total, 74 wheat GH9 genes (TaGH9s) were identified, which were classified into Class A, Class B and Class C and unevenly distributed on chromosomes. We also investigated the gene duplication and reveled that fragments and tandem repeats contributed to the amplification of TaGH9s. TaGH9s had abundant hormone-responsive elements and light-responsive elements, involving JA-ABA crosstalk to regulate anther development. Ten TaGH9s, which highly expressed stamen tissue, were selected to further validate their function in pollen fertility conversion and anther dehiscence. Based on the cell phenotype and the results of the scanning electron microscope at the anther dehiscence period, we found that seven TaGH9s may target miRNAs, including some known miRNAs (miR164 and miR398), regulate the level of cellulose by light and phytohormone and play important roles in pollen fertility and anther dehiscence. Finally, we proposed a hypothesis model to reveal the regulation pathway of TaGH9 on fertility conversion and anther dehiscence. Our study provides valuable insights into the GH9 family in explaining the male sterility mechanism of the wheat photo-thermo-sensitive genetic male sterile (PTGMS) line and generates useful male sterile resources for improving wheat hybrid breeding.

Multifunctional cellulase enzymes are ancestral in Polyneoptera.

Many hemimetabolous insects produce their own cellulase enzymes from the glycoside hydrolase family 9, first observed in termites and cockroaches. Phasmatodea have multiple cellulases, some of which are multifunctional and can degrade xylan or xyloglucan. To discover when these abilities evolved, we identified cellulases from the Polyneoptera sampled by the 1000 Insect Transcriptome and Evolution (1KITE) project, including all cockroach and termite transcriptomes. We hoped to identify what role enzyme substrate specificities had in the evolution of dietary specification, such as leaf-feeding or wood-feeding. Putative cellulases were identified from the transcriptomes and analysed phylogenetically. All cellulases were amplified from an exemplar set of Polyneoptera species using rapid amplification of cDNA ends PCR and heterologously expressed in an insect cell line, then tested against different polysaccharides for their digestive abilities. We identified several multifunctional xyloglucanolytic enzymes across Polyneoptera, plus a large group of cellulase-like enzymes found in nearly all insect orders with no discernible digestive ability. Multifunctional xylanolytic cellulases remain unique to Phasmatodea. The presence or absence of multifunctional enzymes does not impact dietary specification, but rather having multiple, multifunctional cellulase genes is an ancestral state for Polyneoptera and possibly Insecta. The prevalence of multifunctional cellulases in other animals demands further investigation.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Origin, evolution, and divergence of plant class C GH9 endoglucanases.

BACKGROUND: Glycoside hydrolases of the GH9 family encode cellulases that predominantly function as endoglucanases and have wide applications in the food, paper, pharmaceutical, and biofuel industries. The partitioning of plant GH9 endoglucanases, into classes A, B, and C, is based on the differential presence of transmembrane, signal peptide, and the carbohydrate binding module (CBM49). There is considerable debate on the distribution and the functions of these enzymes which may vary in different organisms. In light of these findings we examined the origin, emergence, and subsequent divergence of plant GH9 endoglucanases, with an emphasis on elucidating the role of CBM49 in the digestion of crystalline cellulose by class C members. RESULTS: Since, the digestion of crystalline cellulose mandates the presence of a well-defined set of aromatic and polar amino acids and/or an attributable domain that can mediate this conversion, we hypothesize a vertical mode of transfer of genes that could favour the emergence of class C like GH9 endoglucanase activity in land plants from potentially ancestral non plant taxa. We demonstrated the concomitant occurrence of a GH9 domain with CBM49 and other homologous carbohydrate binding modules, in putative endoglucanase sequences from several non-plant taxa. In the absence of comparable full length CBMs, we have characterized several low strength patterns that could approximate the CBM49, thereby, extending support for digestion of crystalline cellulose to other segments of the protein. We also provide data suggestive of the ancestral role of putative class C GH9 endoglucanases in land plants, which includes detailed phylogenetics and the presence and subsequent loss of CBM49, transmembrane, and signal peptide regions in certain populations of early land plants. These findings suggest that classes A and B of modern vascular land plants may have emerged by diverging directly from CBM49 encompassing putative class C enzymes. CONCLUSION: Our detailed phylogenetic and bioinformatics analysis of putative GH9 endoglucanase sequences across major taxa suggests that plant class C enzymes, despite their recent discovery, could function as the last common ancestor of classes A and B. Additionally, research into their ability to digest or inter-convert crystalline and amorphous forms of cellulose could make them lucrative candidates for engineering biofuel feedstock.

Cellulose degradation in Gastrophysa viridula (Coleoptera: Chrysomelidae): functional characterization of two CAZymes belonging to glycoside hydrolase family 45 reveals a novel enzymatic activity.

Cellulose is a major component of the primary and secondary cell walls in plants. Cellulose is considered to be the most abundant biopolymer on Earth and represents a large potential source of metabolic energy. Yet, cellulose degradation is rare and mostly restricted to cellulolytic microorganisms. Recently, various metazoans, including leaf beetles, have been found to encode their own cellulases, giving them the ability to degrade cellulose independently of cellulolytic symbionts. Here, we analyzed the cellulosic capacity of the leaf beetle Gastrophysa viridula, which typically feeds on Rumex plants. We identified three putative cellulases member of two glycoside hydrolase (GH) families, namely GH45 and GH9. Using heterologous expression and functional assays, we demonstrated that both GH45 proteins are active enzymes, in contrast to the GH9 protein. One GH45 protein acted on amorphous cellulose as an endo-ß-1,4-glucanase, whereas the other evolved to become an endo-ß-1,4-xyloglucanase. We successfully knocked down the expression of both GH45 genes using RNAi, but no changes in weight gain or mortality were observed compared to control insects. Our data indicated that the breakdown of these polysaccharides in G. viridula may facilitate access to plant cell content, which is rich in nitrogen and simple sugars.

Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum.

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.

High-level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants.

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with ß-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.

Genome-wide analysis of the mulberry (Morus abla L.) GH9 gene family and the functional characterization of MaGH9B6 during the development of the abscission zone.

Plant glycoside hydrolase family 9 genes (GH9s) are widely distributed in plants and involved in a variety of cellular and physiological processes. In the current study, nine GH9 genes were identified in the mulberry and were divided into two subfamilies based on the phylogenetic analysis. Conserved motifs and gene structure analysis suggested that the evolution of the two subfamilies is relatively conserved and the glycoside hydrolase domain almost occupy the entire coding region of the GH9s gene. Only segmental duplication has played a role in the expansion of gene family. Collinearity analysis showed that mulberry GH9s had the closest relationship with poplar GH9s. MaGH9B1, MaGH9B6, MaGH9B5, and MaGH9B3 were detected to have transcript accumulation in the stalk of easy-to drop mature fruit drop, suggesting that these could play a role in mulberry fruit drop. Multiple cis-acting elements related to plant hormones and abiotic stress responses were found in the mulberry GH9 promoter regions and showed different activities under exogenous abscisic acid (ABA) and 2,4- dichlorophenoxyacetic acid (2,4-D) stresses. We found that the lignin content in the fruit stalk decreased with the formation of the abscission zone (AZ), which could indirectly reflect the formation process of the AZ. These results provide a theoretical basis for further research on the role of GH9s in mulberry abscission.

Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9.

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Parasitic dodder expresses an arsenal of secreted cellulases with multi-substrate specificity during host invasion.

Cuscuta campestris is a common and problematic parasitic plant which relies on haustoria to connect to and siphon nutrients from host plants. Glycoside hydrolase family 9 (GH9) cellulases (EC 3.2.1.4) play critical roles in plant cell wall biosynthesis and disassembly, but their roles during Cuscuta host invasion remains underexplored. In this study, we identified 22 full-length GH9 cellulase genes in C. campestris genome, which encoded fifteen secreted and seven membrane-anchored cellulases that showed distinct phylogenetic relationships. Expression profiles suggested that some of the genes are involved in biosynthesis and remodeling of the parasite's cell wall during haustoriogenesis, while other genes encoding secreted B- and C-type cellulases are tentatively associated with degrading host cell walls during invasion. Transcriptomic data in a host-free system and in the presence of susceptible or partially resistant tomato hosts, showed for especially GH9B7, GH9B11 and GH9B12 a shift in expression profiles in the presence of hosts, being more highly expressed during host attachment, indicating that Cuscuta can tune cellulase expression in response to a host. Functional analyses of recombinant B- and C-type cellulases showed endoglucanase activities over wide pH and temperature conditions, and activities towards multiple cellulose and hemicellulose substrates. These findings improve our understanding of host cell wall disassembly by Cuscuta, and cellulase activity towards broad substrate range potentially explain its wide host range. This is the first study to provide a broad biochemical insight into Cuscuta GH9 cellulases, which based on our study may have potential applications in industrial bioprocessing.

Characterization of cellulases from softening fruit for enzymatic depolymerization of cellulose.

Cellulose is a major renewable resource for a wide variety of sustainable industrial products. However, for its utilization, finding new efficient enzymes for plant cell wall depolymerization is crucial. In addition to microbial sources, cellulases also exist in plants, however, are less studied. Fleshy fruit ripening includes enzymatic cell wall hydrolysis, leading to tissue softening. Therefore, bilberry (Vaccinium myrtillus L.), which produces small fruits that undergo extensive and rapid softening, was selected to explore cellulases of plant origin. We identified 20 glycoside hydrolase family 9 (GH9) cellulases from a recently sequenced bilberry genome, including four of which showed fruit ripening-specific expression and could be associated with fruit softening based on phylogenetic, transcriptomic and gene expression analyses. These four cellulases were secreted enzymes: two B-types and two C-types with a carbohydrate binding module 49. For functional characterization, these four cellulases were expressed in Pichia pastoris. All recombinant enzymes demonstrated glucanase activity toward cellulose and hemicellulose substrates. Particularly, VmGH9C1 demonstrated high activity and ability to degrade cellulose, xyloglucan, and glucomannan. In addition, all the enzymes retained activity under wide pH (6-10) and temperature ranges (optimum 70 °C), revealing the potential applications of plant GH9 cellulases in the industrial bioprocessing of lignocellulose.

Systematic Analysis of the Grafting-Related Glucanase-Encoding GH9 Family Genes in Pepper, Tomato and Tobacco.

Grafting is an important agricultural practice to control soil-borne diseases, alleviate continuous cropping problems and improve stress tolerance in vegetable industry, but it is relatively less applied in pepper production. A recent study has revealed the key roles of ß-1, 4-glucanase in graft survival. We speculated that the GH9 family gene encoding glucanase may be involved in the obstacles of pepper grafting. Therefore, we performed a systematic analysis of the GH9 family in pepper, tomato and tobacco. A total of 25, 24 and 42 GH9 genes were identified from these three species. Compared with the orthologues of other solanaceous crops, the deduced pepper GH9B3 protein lacks a conserved motif (Motif 5). Promoter cis-element analysis revealed that a wound-responsive element exists in the promoter of tobacco NbGH9B3, but it is absent in the GH9B3 promoter of most solanaceous crops. The auxin-responsive related element is absent in CaGH9B3 promoter, but it presents in the promoter of tobacco, tomato, potato and petunia GH9B3. Tissue and induction expression profiles indicated that GH9 family genes are functionally differentiated. Nine GH9 genes, including CaGH9B3, were detected expressing in pepper stem. The expression patterns of NbGH9B3 and CaGH9B3 in grafting were different in our test condition, with obvious induction in tobacco but repression in pepper. Furthermore, weighted correlation network analysis (WGCNA) revealed 58 transcription factor genes highly co-expressed with NbGH9B3. Eight WRKY binding sites were detected in the promoter of NbGH9B3, and several NbWRKYs were highly co-expressed with NbGH9B3. In conclusion, the missing of Motif 5 in CaGH9B3, and lacking of wound- and auxin-responsive elements in the gene promoter are the potential causes of grafting-related problems in pepper. WRKY family transcription factors could be important regulator of NbGH9B3 in tobacco grafting. Our analysis points out the putative regulators of NbGH9B3, which would be helpful to the functional validation and the study of signal pathways related to grafting in the future.

Interfamily grafting capacity of petunia.

In grafting, an agricultural technique for propagating flower species and fruit trees, two plants are combined to exploit their beneficial characteristics, such as rootstock disease tolerance and vigor. Grafting incompatibility has been observed, however, between distantly related plant combinations, which limits the availability of plant resources. A high grafting capacity has been found in Nicotiana, belonging to Solanaceae, but not in Ipomoea nil, a Convolvulaceae species. Here, we found that Petunia hybrida, another solanaceous species, has similar ability of interfamily grafting, which indicates that interfamily grafting capability in Solanaceae is not limited to the genus Nicotiana. RNA sequencing-based comparative time-series transcriptomic analyses of Nicotiana benthamiana, I. nil, and P. hybrida revealed that N. benthamiana and P. hybrida share a common gene expression pattern, with continued elevated expression of the ß-1,4-glucanase subclade gene GH9B3 observed after interfamily grafting. During self-grafting, GH9B3 expression in each species was similarly elevated, thus suggesting that solanaceous plants have altered regulatory mechanisms for GH9B3 gene expression that allow tissue fusion even with other species. Finally, we tested the effect of the ß-1,4-glucanase inhibitor D-glucono-1,5-lactone, using glucose as a control, on the interfamily grafting usability of P. hybrida with Arabidopsis rootstock. Strong inhibition of graft establishment was observed only with D-glucono-1,5-lactone, thus suggesting the important role of GH9B3 in P. hybrida grafting. The newly discovered grafting compatibility of Petunia with different families enhances the propagation techniques and the production of flower plants.

Turning a potent family-9 free cellulase into an operational cellulosomal component and vice versa.

Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family-9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, whereas its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In this study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C-terminal moiety of Cel9A encompassing the two X2 modules and the family-3b carbohydrate binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family-48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family-9 cellulase.

Insights into the mechanism(s) of digestion of crystalline cellulose by plant class C GH9 endoglucanases.

Biofuels such as γ-valerolactone, bioethanol, and biodiesel are derived from potentially fermentable cellulose and vegetable oils. Plant class C GH9 endoglucanases are CBM49-encompassing hydrolases that cleave the ß (1 → 4) glycosidic linkage of contiguous D-glucopyranose residues of crystalline cellulose. Here, I analyse 3D-homology models of characterised and putative class C enzymes to glean insights into the contribution of the GH9, linker, and CBM49 to the mechanism(s) of crystalline cellulose digestion. Crystalline cellulose may be accommodated in a surface groove which is imperfectly bounded by the GH9_CBM49, GH9_linker, and linker_CBM49 surfaces and thence digested in a solvent accessible subsurface cavity. The physical dimensions and distortions thereof, of the groove, are mediated in part by the bulky side chains of aromatic amino acids that comprise it and may also result in a strained geometry of the bound cellulose polymer. These data along with an almost complete absence of measurable cavities, along with poorly conserved, hydrophobic, and heterogeneous amino acid composition, increased atomic motion of the CBM49_linker junction, and docking experiements with ligands of lower degrees of polymerization suggests a modulatory rather than direct role for CBM49 in catalysis. Crystalline cellulose is the de facto substrate for CBM-containing plant and non-plant GH9 enzymes, a finding supported by exceptional sequence- and structural-homology. However, despite the implied similarity in general acid-base catalysis of crystalline cellulose, this study also highlights qualitative differences in substrate binding and glycosidic bond cleavage amongst class C members. Results presented may aid the development of novel plant-based GH9 endoglucanases that could extract and utilise potential fermentable carbohydrates from biomass. Graphical Abstract Crystalline cellulose digestion by plant class C GH9 endoglucanases - an in silico assessment of function.

Overproduction of native endo-ß-1,4-glucanases leads to largely enhanced biomass saccharification and bioethanol production by specific modification of cellulose features in transgenic rice.

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-ß-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 ß-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 ß-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.

Restoration of cellulase activity in the inactive cellulosomal protein Cel9V from Ruminiclostridium cellulolyticum.

Ruminiclostridium cellulolyticum produces extracellular cellulosomes which contain interalia numerous family-9 glycoside hydrolases, including the inactive Cel9V. The latter shares the same organization and 79% sequence identity with the active cellulase Cel9E. Nevertheless, two aromatic residues and a four-residue stretch putatively critical for the activity are missing in Cel9V. Introduction of one Trytophan and the four-residue stretch restored some weak activity in Cel9V, whereas the replacement of its catalytic domain by that of Cel9E generated a fully active cellulase. Altogether our data indicate that a series of mutations in the catalytic domain of Cel9V lead to an essentially inactive cellulase.

Genome sequence of Rhizomucor pusillus FCH 5.7, a thermophilic zygomycete involved in plant biomass degradation harbouring putative GH9 endoglucanases.

We report here the annotated draft genome sequence of the thermophilic zygomycete Rhizomucor pusillus strain FCH 5.7, isolated from compost soil in Vietnam. The genome assembly contains 25.59 Mb with an overall GC content of 44.95%, and comprises 10,898 protein coding genes. Genes encoding putative cellulose-, xylan- and chitin-degrading proteins were identified, including two putative endoglucanases (EC 3.2.1.4) from glycoside hydrolase family 9, which have so far been mostly assigned to bacteria and plants.