Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.
ABSTRACT: Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have ?-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-?-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against ?-(1,5)-linked l-Ara (LM6) and ?-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.
Project description:Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.
Project description:Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 ?-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave ?-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl ?-l-arabinofuranoside were also detected. After selective removal of ?-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining ?-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove ?-l-Araf residues that are (1?2) and (1?3) linked to monosubstituted ?-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed ?-propeller fold and revealed a molecular Velcro in blade V between the ?1 and ?21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the ? propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.
Project description:The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, ?-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited ?-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-?-d-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, ?-l-Araf-(1?2)-d-Xylp or ?-l-Araf-(1?3)-d-Xylp and ?-l-Araf-(1?2)-[?-l-Araf-(1?3)]-?-d-Xylp Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, ?-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.
Project description:Background:The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling. Results:Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R2/R2cv/R2ev and RPD values for a perfect prediction capacity. Conclusions:Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.
Project description:Cellulosic ethanol is an alternative for increasing the amount of bioethanol production in the world. In Brazil, sugarcane leads the bioethanol production, and to improve its yield, besides bagasse, sugarcane straw is a possible feedstock. However, the process that leads to cell wall disassembly under field conditions is unknown, and understanding how this happens can improve sugarcane biorefinery and soil quality. In the present work, we aimed at studying how sugarcane straw is degraded in the field after 3, 6, 9, and 12 months. Non-structural and structural carbohydrates, lignin content, ash, and cellulose crystallinity were analyzed. The cell wall composition was determined by cell wall fractionation and determination of monosaccharide composition. Non-structural carbohydrates degraded quickly during the first 3 months in the field. Pectins and lignin remained in the plant waste for up to 12 months, while the hemicelluloses and cellulose decreased 7.4 and 12.4%, respectively. Changes in monosaccharide compositions indicated solubilization of arabinoxylan (xylose and arabinose) and β-glucans (β-1,3 1,4 glucan; after 3 months) followed by degradation of cellulose (after 6 months). Despite cellulose reduction, the xylose:glucose ratio increased, suggesting that glucose is consumed faster than xylose. The degradation and solubilization of the cell wall polysaccharides concomitantly increased the level of compounds related to recalcitrance, which led to a reduction in saccharification and an increase in minerals and ash contents. Cellulose crystallinity changed little, with evidence of silica at the latter stages, indicating mineralization of the material. Our data suggest that for better soil mineralization, sugarcane straw must stay in the field for over 1 year. Alternatively, for bioenergy purposes, straw should be used in less than 3 months.
Project description: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-?-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 ?-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 ?-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.
Project description:<h4>Background</h4>Rice not only produces grains for human beings, but also provides large amounts of lignocellulose residues, which recently highlighted as feedstock for biofuel production. Genetic modification of plant cell walls can potentially enhance biomass saccharification; however, it remains a challenge to maintain a normal growth with enhanced lodging resistance in rice.<h4>Results</h4>In this study, rice (<i>Oryza sativa</i>) mutant <i>fc17</i>, which harbors the substitution (F426S) at the plant-conserved region (P-CR) of cellulose synthase 4 (CESA4) protein, exhibited slightly affected plant growth and 17% higher lodging resistance compared to the wild-type. More importantly, the mutant showed a 1.68-fold enhancement in biomass saccharification efficiency. Cell wall composition analysis showed a reduction in secondary wall thickness and cellulose content, and compensatory increase in hemicelluloses and lignin content. Both X-ray diffraction and calcofluor staining demonstrated a significant reduction in cellulose crystallinity, which should be a key factor for its high saccharification. Proteomic profiling of wild-type and <i>fc17</i> plants further indicated a possible mechanism by which mutation induces cellulose deposition and cell wall remodeling.<h4>Conclusion</h4>These results suggest that CESA4 P-CR site mutation affects cell wall features especially cellulose structure and thereby causes enhancement in biomass digestion and lodging resistance. Therefore, CESA4 P-CR region is promising target for cell wall modification to facilitate the breeding of bioenergy rice.
Project description:The major plant sugar l-arabinose (l-Ara) has two different ring forms, l-arabinofuranose (l-Araf) and l-arabinopyranose (l-Arap). Although l-Ara mainly appears in the form of ?-l-Araf residues in cell wall components, such as pectic ?-1,3:1,5-arabinan, arabinoxylan, and arabinogalactan-proteins (AGPs), lesser amounts of it can also be found as ?-l-Arap residues of AGPs. Even though AGPs are known to be rapidly metabolized, the enzymes acting on the ?-l-Arap residues remain to be identified. In the present study, four enzymes, which we call ?-l-ARAPASE (APSE) and ?-GALACTOSIDASE 1 (AGAL1), AGAL2, and AGAL3, are identified as those enzymes that are likely to be responsible for the hydrolysis of the ?-l-Arap residues in Arabidopsis thaliana. An Arabidopsis apse-1 mutant showed significant reduction in ?-l-arabinopyranosidase activity, and an apse-1 agal3-1 double-mutant exhibited even less activity. The apse-1 and the double-mutants both had more ?-l-Arap residues in the cell walls than wild-type plants. Recombinant APSE expressed in the yeast Pichia pastoris specifically hydrolyzed ?-l-Arap residues and released l-Ara from gum arabic and larch arabinogalactan. The recombinant AGAL3 also showed weak ?-l-arabinopyranosidase activity beside its strong ?-galactosidase activity. It appears that the ?-l-Arap residues of AGPs are hydrolysed mainly by APSE and partially by AGALs in Arabidopsis.
Project description:<h4>Background</h4>Wheat and rice are important food crops with enormous biomass residues for biofuels. However, lignocellulosic recalcitrance becomes a crucial factor on biomass process. Plant cell walls greatly determine biomass recalcitrance, thus it is essential to identify their key factors on lignocellulose saccharification. Despite it has been reported about cell wall factors on biomass digestions, little is known in wheat and rice. In this study, we analyzed nine typical pairs of wheat and rice samples that exhibited distinct cell wall compositions, and identified three major factors of wall polymer features that affected biomass digestibility.<h4>Results</h4>Based on cell wall compositions, ten wheat accessions and three rice mutants were classified into three distinct groups each with three typical pairs. In terms of group I that displayed single wall polymer alternations in wheat, we found that three wall polymer levels (cellulose, hemicelluloses and lignin) each had a negative effect on biomass digestibility at similar rates under pretreatments of NaOH and H2SO4 with three concentrations. However, analysis of six pairs of wheat and rice samples in groups II and III that each exhibited a similar cell wall composition, indicated that three wall polymer levels were not the major factors on biomass saccharification. Furthermore, in-depth detection of the wall polymer features distinctive in rice mutants, demonstrated that biomass digestibility was remarkably affected either negatively by cellulose crystallinity (CrI) of raw biomass materials, or positively by both Ara substitution degree of non-KOH-extractable hemicelluloses (reverse Xyl/Ara) and p-coumaryl alcohol relative proportion of KOH-extractable lignin (H/G). Correlation analysis indicated that Ara substitution degree and H/G ratio negatively affected cellulose crystallinity for high biomass enzymatic digestion. It was also suggested to determine whether Ara and H monomer have an interlinking with cellulose chains in the future.<h4>Conclusions</h4>Using nine typical pairs of wheat and rice samples having distinct cell wall compositions and wide biomass saccharification, Ara substitution degree and monolignin H proportion have been revealed to be the dominant factors positively determining biomass digestibility upon various chemical pretreatments. The results demonstrated the potential of genetic modification of plant cell walls for high biomass saccharification in bioenergy crops.
Project description:Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of ?-1,4-linked xylose residues with substitutions that include ?-(1?2)-linked glucuronosyl, 4-O-methyl glucuronosyl, and ?-1,2- and ?-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by (1)H-NMR to be (?-1,4-Xyl)(4) with a ?-Xylp-(1?2)-?-Araf-(1?3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation.