RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

Isolation and characterization of a Clostridium sp with cinnamoyl esterase activity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis.

Microbiology 154 (2008) 2719-2729

In vitro fermentation by human fecal microflora of wheat arabinoxylans

The fermentation of three arabinoxylan (AX) fractions from wheat by the human fecal microflora was investigated in vitro. Three AX fractions, with average molecular masses of 354, 278, and 66 kDa, were incorporated into miniature-scale batch cultures (with inulin as a positive prebiotic control) with feces from three healthy donors, aged 23-29. Microflora changes were monitored by the culture-independent technique, fluorescent in situ hybridization, and short chain fatty acid (SCFA) and lactic acid production were measured by high-performance liquid chromatography. Total cell numbers increased significantly in all treated cultures, and the fermentation of AX was associated with a proliferation of the bifidobacteria, lactobacilli, and eubacteria groups. Smaller but statistically significant increases in bacteroides and clostridia groups were also observed. All AX fractions had comparable bifidogenic impacts on the microflora at 5 and 12 h, but the 66 kDa AX was particularly selective for lactobacilli. Eubacteria increased significantly on all AX fractions, particularly on 66 kDa AX. As previously reported, inulin gave a selective increase in bifidobacteria. All supplemented cultures showed significant rises in total SCFA production, with a particularly high proportion of butyric acid being produced from AX fermentation. The prebiotic effect, that is, the selectivity of AX for bifidobacteria and lactobacilli groups, increased as the molecular mass of the AX decreased. This suggests that molecular mass may influence the fermentation of AX in the colon.

Milling performance of north European hull-less barleys and characterization of resultant millstreams

Four hull-less barley samples were milled on a Buhler MLU 202 laboratory mill and individual and combined milling fractions were characterized. The best milling performance was obtained when the samples were conditioned to 14.3% moisture. Yields were 37-48% for straight-run flour, 47-56% for shorts, and 5-8% for bran. The beta-glucan contents of the straight-run white flours were 1.6-2.1%, of which approximate to49% was water-extractable. The arabinoxylan contents were 1.2-1.5%, of which approximate to17% was water-extractable. Shorts and bran fractions contained more beta-glucan (4.2-5.8% and 3.0-4.7%, respectively) and arabinoxylan (6.1-7.7% and 8.1-11.8%, respectively) than the white flours. For those fractions, beta-glucan extractability was high (58.5 and 52.3%, respectively), whereas arabinoxylan extractability was very low (approximate to6.5 and 2.0%, respectively). The straight-run white flours had low alpha-amylase, beta-glucanase, and endoxylanase activities. The highest alpha-amylase activity was found in the shorts fractions and the highest beta-glucanase and endoxylanase activities were generally found in the bran fractions. Endoxylanase inhibitor activities were low in the white flours and highest in the shorts fractions. High flavanoid, tocopherol, and tocotrienol contents were found in bran and shorts fractions.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by alpha-(1,2)- and alpha-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases alpha-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for alpha-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

A randomised, double-blind, placebo controlled cross-over study to determine the gastrointestinal effects of consumption of arabinoxylanoligosaccharides enriched bread in healthy volunteers

BACKGROUND: Prebiotics are food ingredients, usually non-digestible oligosaccharides, that are selectively fermented by populations of beneficial gut bacteria. Endoxylanases, altering the naturally present cereal arabinoxylans, are commonly used in the bread industry to improve dough and bread characteristics. Recently, an in situ method has been developed to produce arabinoxylan-oligosaccharides (AXOS) at high levels in breads through the use of a thermophilic endoxylanase. AXOS have demonstrated potentially prebiotic properties in that they have been observed to lead to beneficial shifts in the microbiota in vitro and in murine, poultry and human studies. METHODS: A double-blind, placebo controlled human intervention study was undertaken with 40 healthy adult volunteers to assess the impact of consumption of breads with in situ produced AXOS (containing 2.2 g AXOS) compared to non-endoxylanase treated breads. Volatile fatty acid concentrations in faeces were assessed and fluorescence in situ hybridisation was used to assess changes in gut microbial groups. Secretory immunoglobulin A (sIgA) levels in saliva were also measured. RESULTS: Consumption of AXOS-enriched breads led to increased faecal butyrate and a trend for reduced iso-valerate and fatty acids associated with protein fermentation. Faecal levels of bifidobacteria increased following initial control breads and remained elevated throughout the study. Lactobacilli levels were elevated following both placebo and AXOS-breads. No changes in salivary secretory IgA levels were observed during the study. Furthermore, no adverse effects on gastrointestinal symptoms were reported during AXOS-bread intake. CONCLUSIONS: AXOS-breads led to a potentially beneficial shift in fermentation end products and are well tolerated.

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production. © 2012 Springer-Verlag Berlin Heidelberg.

Effect of a thermoascus aurantiacus thermostable enzyme cocktail on wheat bread quality

Thermophilic fungus Thermoascus aurantiacus (CBMAI 756) on solid-state fermentation using corncob as a nutrient source produces an enzyme pool with the potential to be used in bread making. In this paper, the use of this enzyme cocktail as a wheat bread improver was reported. Both products released by flour arabinoxylan degradation and bread quality were investigated. The main product released through enzyme activity after prolonged incubation was xylose indicating the presence of xylanase; however, a small amount of xylobiose and arabinose also confirmed the presence of xylosidase and α-L- arabinofuranosidase, respectively. Enzyme mixture in vitro mainly attacked water-unextractable arabinoxylan contributing to beneficial effect in bread making. The use of an optimal enzyme concentration (35 U xylanase/100 g of flour) increased specific volume (22%), reduced crumb firmness (25%), and reduced amylopectin retrogradation (17%) during bread storage. In conclusion, the enzyme cocktail produced by T. aurantiacus CBMAI 756 can improve wheat bread quality. © 2013 Elsevier Ltd.

Aplicação de xilanase e/ou ciclodextria glicotransferase (CGTase) na produção de pães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding a-L-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0-5.0) and an optimum temperature of 70 degrees C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 degrees C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. (C) 2012 Elsevier Ltd. All rights reserved.

Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.