Glycoside hydrolases (GHs) are enzymes that are able to rearrange the plant cell wall polysaccharides, being developmental- and stress-regulated. Such proteins are used in enzymatic cocktails for biomass hydrolysis in the second-generation ethanol (E2G) production. In this chapter, we investigate GHs identified in plant cell wall proteomes by predicting their functions through alignment with homologous plant and microorganism sequences and identification of functional domains. Up to now, 49 cell wall GHs were identified in sugarcane and 114 in Brachypodium distachyon. We could point at candidate proteins that could be targeted to lower biomass recalcitrance. We more specifically addressed several GHs with predicted cellulase, hemicellulase, and pectinase activities, such as β-xylosidase, α and β-galactosidase, α-N-arabinofuranosidases, and glucan β-glucosidases. These enzymes are among the most used in enzymatic cocktails to deconstruct plant cell walls. As an example, the fungi arabinofuranosidases belonging to the GH51 family, which were also identified in sugarcane and B. distachyon, have already been associated to the degradation of hemicellulosic and pectic polysaccharides, through a peculiar mechanism, probably more efficient than other GH families. Future research will benefit from the information available here to design plant varieties with self-disassembly capacity, making the E2G more cost-effective through the use of more efficient enzymes.
- Brachypodium distachyon
- cell wall proteomes
- glycoside hydrolase (GH)
- second-generation ethanol
The production of second-generation ethanol (E2G) provides an additional source of energy in the sugar and ethanol sector by increasing the biofuel yield without expanding the crop area, thus leading to a sustainable production system. However, for the process to become financially competitive in comparison with first-generation ethanol (E1G), it is necessary to reduce the costs related to the lignocellulosic biomass processing required to recover and break the sugars present in plant cell walls . The principal barrier to the conversion of lignocellulosic biomass into bioethanol or chemicals is the insoluble lignin network that surrounds and shields the cellulose microfibrils from degrading enzymes. The high energy and environmental costs of the treatments necessary to overcome these drawbacks constitute major hurdles to commercial E2G production . To overcome these limitations, many studies have focused on the identification of enzymes related to biomass pretreatment and hydrolysis processes. The majority of the enzymes that compose enzymatic cocktails are proteins prospected from fungi that belong to different glycoside hydrolase (GH) families .
The raw material for E2G production is plant fiber, which is mainly composed of cell walls. Nevertheless, less work has been devoted to study plant cell wall proteins (CWPs), and thus, to understand how the plant mechanisms themselves function to loosen and tighten up back the cell wall in order to promote cell growth and adapt to their changing environment. Accordingly, a common opinion today is that it is important to understand how cell walls are built up to improve the biomass deconstruction processes [4, 5].
Plant cells are surrounded by a wall characterized by its specific structure and composition . Cell walls are mainly composed of polysaccharides, lignin, suberin, waxes, proteins, calcium, boron, and water, and have the ability to self-assemble . Plants have two different kinds of cell wall deposition. Primary cell walls are synthesized in still-growing cells, whose form is not definitive, and thus, they can undergo growth and expansion. Secondary cell walls are synthesized in already fully expanded cells which are differentiating to perform specific functions, like xylem and fibers cells. In lignified secondary walls, the proportion of cellulose is higher than in primary cell walls with a higher degree of polymerization and crystallinity specificities . Cellulose, hemicellulose, and pectins are the cell wall polysaccharides, and the biogenesis of cell walls involves their synthesis in intracellular compartments or at the plasma membrane, secretion, assembly, and rearrangement
Displaying roles in cell growth, enzymes are part of the cell wall proteome. Glycosidases and glycanases have exo- and endo-GH activities, respectively, while trans-glycosidases and trans-glycanases perform exo- and endo-transglycosylation, respectively. Pectin methylesterases and pectin acetylesterases control the degree of homogalacturonan methylesterification and acetylation, respectively . Class III peroxidases (Prxs) can either form covalent bonds by oxidizing aromatic compounds such as monolignols or aromatic amino acids or produce reactive oxygen species that participate in non-enzymatic breakage of covalent bonds of polysaccharides . All these proteins belong to multigene families and their genes are differentially regulated during plant development and in response to environmental changes.
GHs are of special interest, since they can hydrolyze the glycosidic bonds from two carbohydrates or from a carbohydrate and a non-carbohydrate moiety, thus actively contributing to cell wall polymer rearrangements. In
In this chapter, we will describe cell wall GHs identified in the cell wall proteomes of sugarcane and
2. Plant GH families
In plants, several strategies have been used in order to extract and identify CWPs with a high number of GHs, such as vacuum-infiltration protocol with saline solution and identification of proteins predicted by bioinformatics to be targeted to the secretory pathway [23, 24, 25]. In
Conversely, in the monocot rice, GH17 is the one that presents the highest number of members, followed by GH28 . The cell wall particularities of dicots (e.g.,
By bioinformatic analyses of amino acid sequences, it is possible to classify newly identified GHs into families. We have done it for the GHs identified in the sugarcane and the
3. GHs identified in sugarcane and
B. distachyoncell wall proteomes
In sugarcane, 49 GHs have been identified in cell wall proteomes [28, 29]. They are distributed in 16 GH families. The GH3 family is the best represented (~20%), followed by GH17 (~16%), GH18 (~12%), and GH1 (~8%) (Figure 1). This distribution varies according to organ and developmental stage. In cell suspension cultures, only 4 GH families were identified among which GH3 was the most populated . In 2-month-old stems, 7 GH families were found, GH3 also being the most represented . Leaves recovered few GHs, from families 19, 27 (young leaves only), 28, and 31 (young leaves only). Apical internodes mainly contained GH3 members, whereas mostly GH17 members were found in basal ones . Noteworthy, it should be mentioned that the absence of some GH families in a given cell wall proteomes could be due to technical limitations or differential accessibility as a consequence of differences in cell wall structure.
The large size of the GH1, GH17, and GH28 families is probably linked to their roles in the assembly and in the rearrangement of cell wall polysaccharides . Usually the GH1, GH16, GH17, and GH35 families are less represented in dicots than in monocots . GH17 display glucan-1,3-β-glucosidase activity and possible substrates could be mixed (1,3)(1,4)-β-D-glucans . This is consistent with the fact that only type II grass cell walls present this kind of hemicellulose.
After a survey of the cell wall proteomes described so far and collecting information regarding microorganism enzymes used for biomass deconstruction, we decided to focus this review on the GH1, GH3, GH17, GH27, GH35, and GH51 families. We have predicted functional and structural domains in newly identified CWPs using the PredictProtein bioinformatic software and grouping them in families . Since plant cells perform cell expansion themselves by involving cell wall polysaccharide rearrangements, the plant mechanisms could be mimicked by the enzymes used in cocktails. The comparison of plant and microorganisms enzymes presently used for biomass hydrolysis could contribute to determining their common characteristics and which specificities of plant enzymes could be copied in order to improve industrial cell wall deconstruction processes. Conversely, this comparative study could help in identifying which of the characteristics of microorganism enzymes could be engineered in plant species in order to obtain biomass with less recalcitrant cell walls.
4. GH1, GH3, and GH51
The GH1 family mainly comprises β-glucosidases, which are found in several organisms performing different functions. In plants, they are involved in cell wall catabolism, signaling, lignification, defense, symbiosis, and secondary metabolism. Putative β-glucosidase genes have been shown to be induced by biotic and abiotic stresses and they were considered critical for the success of plant development in stressful environments [36, 37, 38, 39, 40]. Accordingly, plants are the organisms that have the highest number of GH1s, e.g., 48 in
The plant cell wall is a large polysaccharide repository that contains a large amount of glucosyl residues. β-glucosidases play important roles in cell wall formation and plant development, because they participate in cell wall polysaccharide turnover . In sugarcane  and
Ten GH3s have been identified in sugarcane [28, 29, 30] and nine GH3s have been identified in
The fungi β-glucosidases can degrade cellulose together with other kinds of enzymes, like endoglucanases and cellobiohydrolases. They separate the molecules of glucose from cellobiose, thus being used in enzymatic cocktails to produce cellulosic bioethanol . In barley, the structure of the GH3 β-D-glucan exohydrolase ExoI was determined through X-ray crystallography, showing a two-domain globular structure being different from that of GH1 . Besides the catalytic site, this enzyme has another binding site for (1 → 3, 1 → 4)-β-D-glucans only identified in monocots. Xylan 1,4-β-D-xylosidases hydrolyze xylose from xylo-oligosaccharides. These enzymes have several uses, such as in the industrial processes related to bread dough, animal feed digestibility, and deinking of recycled papers. In enzymatic cocktails, they are considered the most efficient enzymes to break glycosidic bonds of hemicelluloses . The few GH51 members identified in sugarcane and
To compare plant and microorganism GH1 and GH3, we have performed phylogenic analyses. Some GH1 and GH3 identified in cell wall proteomes of sugarcane and
Finally, the tree is split into two distinct clades, each containing either one or two closely related
5. GH 17
GH17 are encoded by large gene families in plants. In
β-1,3-glucanases have been shown to be important proteins involved in plant defense reactions against pathogens and are considered as pathogenesis-related proteins of the PR-2 family . Their role is hydrolysis of the β-1,3-glucan bonds, an important structural component of fungal cell walls, resulting in their destabilization and in the release of elicitors that further stimulate defense responses . This antifungal activity was shown both
According to phylogenic analyses, the GH17 family is divided into three distinct clades (denoted α, β, and γ) [61, 62], with 10% of its members having cell wall-related functions . GH17 of the α clade are more related to stem elongation, but also responsive to gibberellin, those of the β and γ clades are more related to stress response and defense against pathogens [62, 63, 64, 65]. In addition to the GH17 domain
Other studies also revealed the antifungal effects of plant extracellular chitinases (GH18 and GH19) in combination with those of GH17 . Indeed, fungi cell walls are composed of chitin and of branched β-(1,3):β-(1,6) glucans [57, 70, 71, 72, 73]. Thereby, transgenic plants overexpressing a chitinase and/or a ß-l,3 glucanase became less susceptible to fungal attack [74, 75].
6. GH27 and GH35
The GH27 identified in cell wall proteomes of both sugarcane and
GH35 are mainly β-galactosidases (EC 184.108.40.206), but exo-1,4-β-D-glucosaminidase (E.C 220.127.116.11) and exo-β-1,4-galactanases (EC 3.2.1.-) also belong to this family. β-galactosidases are found in microorganisms such as bacteria, fungi, and yeast, as well as in animals and plants . They catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in different molecules, like glycoproteins, oligosaccharides, glycolipids and lactose (www.cazy.org). β -galactosidases are classified in two families: GH2 are predominantly found in microorganisms (around 70%), and GH35 are found in plants [78, 79].
GH35 can be distributed into two main groups according to their preferred substrates: hydrolysis of pectic β-1,4-galactans, cleavage of β-1,3- and β-1,6-galactosyl linkages of
7. Concluding remarks
Microorganisms use an arsenal of GHs to degrade plant cell walls, in order to establish themselves in their host. Similar mechanisms are thought to be used in their own plant cell wall modification, since plant cell walls embrace several types of carbohydrates with a variety of structures and biological functions. For sugarcane biomass deconstruction, the first step proposed is the use of pectinases to release pectins, such as endopolygalacturonases, AFases, and β-galactosidases, along with pectin methylesterases. Lichenases are used to hydrolyze mixed linked β-D-glucan. The remaining polymers, cellulose, and hemicelluloses, would have to be treated with a mixture of enzymes like endo-β-xylanases, α-arabinofuranosidases, xyloglucanases, α-xylosidases, and β-galactosidases. Finally, cellulose could be the substrate of endo-β-glucanases, cellobiohydrolases, and β-glucosidases .
Besides many studies focusing on microorganism enzymes to optimize E2G production, this work has evaluated the plant enzymes that are assumed to display similar activities. Since plant GHs perform cell wall breakage and expansion, a deeper investigation of their structure could be performed in order to produce more efficient chimeric enzymes to be used in enzymatic cocktails. It is difficult to establish GH functions from their amino acid sequences because proteins from the same GH family may have diverse substrates and roles . However, we were able to predict functions for the GHs identified in the cell wall proteomes of sugarcane and
Therefore, this work has contributed to provide target proteins that could possibly be used in future research to facilitate cheaper E2G production, besides allowing a more detailed analysis of the cell wall proteomes of the grasses, sugarcane and
The authors thank Dr. Roberto Ruller for the microorganism suggestions.