Mechanistic insights from plant heteromannan synthesis in yeast.

Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of ß-(1-4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.

Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus.

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme-substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme-substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.

Aspergillus fumigatus Mnn9 is responsible for mannan synthesis and required for covalent linkage of mannoprotein to the cell wall.

Owing to the essential role in protection of the Aspergillus fumigatus cell against human defense reactions, its cell wall has long been taken as a promising antifungal target. The cell wall of A. fumigatus composed of chitin, glucan and galactomannan and mannoproteins. Although galactomannan has been used as a diagnostic target for a long time, its biosynthesis remains unknown in A. fumigatus. In this study, a putative α1,6-mannosyltransferase gene mnn9 was identified in A. fumigatus. Deletion of the mnn9 gene resulted in an increased sensitivity to calcofluor white, Congo red, or hygromycin B as well as in reduced cell wall components and abnormal polarity. Although there was no major effect on N-glycan synthesis, covalently-linked cell wall mannoprotein Mp1 was significantly reduced in the mutant. Based on our results, we propose that Mnn9p is a mannosyltransferase responsible for the formation of the α-mannan in cell wall mannoproteins, potentially via elongation of O-linked mannose chains.

The cell envelope-associated phospholipid-binding protein LmeA is required for mannan polymerization in mycobacteria.

The integrity of the distinguishing, multilaminate cell envelope surrounding mycobacteria is critical to their survival and pathogenesis. The prevalence of phosphatidylinositol mannosides in the cell envelope suggests an important role in the mycobacterial life cycle. Indeed, deletion of the pimE gene (ΔpimE) encoding the first committed step in phosphatidylinositol hexamannoside biosynthesis in Mycobacterium smegmatis results in the formation of smaller colonies than wild-type colonies on Middlebrook 7H10 agar. To further investigate potential contributors to cell-envelope mannan biosynthesis while taking advantage of this colony morphology defect, we isolated spontaneous suppressor mutants of ΔpimE that reverted to wild-type colony size. Of 22 suppressor mutants, 6 accumulated significantly shorter lipomannan or lipoarabinomannan. Genome sequencing of these mutants revealed mutations in genes involved in the lipomannan/lipoarabinomannan biosynthesis, such as those encoding the arabinosyltransferase EmbC and the mannosyltransferase MptA. Furthermore, we identified three mutants carrying a mutation in a previously uncharacterized gene, MSMEG_5785, that we designated lmeA Complementation of these suppressor mutants with lmeA restored the original ΔpimE phenotypes and deletion of lmeA in wild-type M. smegmatis resulted in smaller lipomannan, as observed in the suppressor mutants. LmeA carries a predicted N-terminal signal peptide, and density gradient fractionation and detergent extractability experiments indicated that LmeA localizes to the cell envelope. Using a lipid ELISA, we found that LmeA binds to plasma membrane phospholipids, such as phosphatidylethanolamine and phosphatidylinositol. LmeA is widespread throughout the Corynebacteriales; therefore, we concluded that LmeA is an evolutionarily conserved cell-envelope protein critical for controlling the mannan chain length of lipomannan/lipoarabinomannan.

Genome-wide discovery of tissue-specific miRNAs in clusterbean (Cyamopsis tetragonoloba) indicates their association with galactomannan biosynthesis.

Owing to the presence of 80% soluble dietary fibre, high protein content and high value gum, clusterbean (Cyamopsis tetragonoloba) has recently emerged as an economically important legume. The developing clusterbean seeds accumulate 90% galactomannans in the endosperm and, therefore, can be used as a model crop to understand galactomannan biosynthesis and its regulation. miRNAs are tiny master regulators of their corresponding target genes, resulting in variations in the amounts of their metabolic end products. To understand the role of these regulators in galactomannan biosynthesis regulation, small RNA libraries were prepared and sequenced from five tissues of clusterbean genotype RGC-936, and miRanalyzer and DSAP programs were used to identify conserved miRNAs and novel small RNAs. A total of 187 known and 171 novel miRNAs were found to be differentially expressed, of which 10 miRNAs were validated. A complicated network topology and 35% sharing of the target mRNAs between known and novel miRNAs suggest random evolution of novel miRNAs. The gene ontology (GO) annotation of potential target genes revealed the genes coding for signalling and carbohydrate metabolism (50.10%), kinases and other enzymes (20.75%), transcription factors (10.20%), transporters (8.35%) and other targets (10.6%). Two novel unigenes were annotated as ManS (mannosyltransferase/mannan synthase) and UGE (UDP- D-glucose 4-epimerase) and validated as targets for three novel miRNAs, that is Ct-miR3130, Ct-miR3135 and Ct-miR3157. Our findings reveal that these novel miRNAs could play an important role in the regulation of the galactomannan pathway in C. tetragonoloba and possibly other galactomannan-producing species.

Biosynthesis of galactomannans found in filamentous fungi belonging to Pezizomycotina.

The galactomannans (GMs) that are produced by filamentous fungi belonging to Pezizomycotina, many of which are pathogenic for animals and plants, are polysaccharides consisting of α-(1→2)-/α-(1→6)-mannosyl and ß-(1→5)-/ß-(1→6)-galactofuranosyl residues. GMs are located at the outermost layer of the cell wall. When a pathogenic fungus infects a host, its cell surface must be in contact with the host. The GMs on the cell surface may be involved in the infection mechanism of a pathogenic fungus or the defense mechanism of a host. There are two types of GMs in filamentous fungi, fungal-type galactomannans and O-mannose type galactomannans. Recent biochemical and genetic advances have facilitated a better understanding of the biosynthesis of both types. This review summarizes our current information on their biosynthesis.

Insights from NMR Spectroscopy into the Conformational Properties of Man-9 and Its Recognition by Two HIV Binding Proteins.

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.

Cell wall polysaccharides released during the alcoholic fermentation by Schizosaccharomyces pombe and S. japonicus: quantification and characterization.

The present work demonstrates that yeasts belonging to the Schizosaccharomyces genus release a high quantity of polysaccharides of cell wall origin starting from the onset of the alcoholic fermentation. By the end of the alcoholic fermentation, all of the Schizosaccharomyces yeast strains released a quantity of polysaccharides approximately 3-7 times higher than that released by a commercial Saccharomyces cerevisiae yeast strain under the same fermentative conditions of synthetic juice. A higher content of polysaccharide was found in media fermented by Schizosaccharomyces japonicus with respect to that of Schizosaccharomyces pombe. Some of the strains evaluated were also able to produce high levels of pyruvic acid, which has been shown to be an important compound for color stability of wine. The presence of strains with different malic acid consumption patterns along with high polysaccharide release would enable production of naturally modified wines with enhanced mouth feel and reduced acidity. The chemical analysis of the released polysaccharides demonstrated divergence between the two yeast species S. pombe and S. japonicus. A different mannose/galactose ratio and a different percentage of proteins was observed on the polysaccharides released by S. pombe as compared to S. japonicus. Analysis of the proteins released in the media revealed the presence of a glycoprotein with a molecular size around 32-33 kDa only for the species S. japonicus. Mass spectrometry analysis of carbohydrate moieties showed similar proportions among the N-glycan chains released in the media by both yeast species but differences between the two species were also observed. These observations suggest a possible role of rapid MALDI-TOF screening of N-glycans compositional fingerprint as a taxonomic tool for this genus. Polysaccharides release in the media, in particular galactomannoproteins in significant amounts, could make these yeasts particularly interesting also for the industrial production of exogenous polysaccharide preparations.

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage.

Plants invest a lot of their resources into the production of an extracellular matrix built of polysaccharides. While the composition of the cell wall is relatively well characterized, the functions of the individual polymers and the enzymes that catalyze their biosynthesis remain poorly understood. We exploited the Arabidopsis (Arabidopsis thaliana) seed coat epidermis (SCE) to study cell wall synthesis. SCE cells produce mucilage, a specialized secondary wall that is rich in pectin, at a precise stage of development. A coexpression search for MUCILAGE-RELATED (MUCI) genes identified MUCI10 as a key determinant of mucilage properties. MUCI10 is closely related to a fenugreek (Trigonella foenumgraecum) enzyme that has in vitro galactomannan α-1,6-galactosyltransferase activity. Our detailed analysis of the muci10 mutants demonstrates that mucilage contains highly branched galactoglucomannan (GGM) rather than unbranched glucomannan. MUCI10 likely decorates glucomannan, synthesized by CELLULOSE SYNTHASE-LIKE A2, with galactose residues in vivo. The degree of galactosylation is essential for the synthesis of the GGM backbone, the structure of cellulose, mucilage density, as well as the adherence of pectin. We propose that GGM scaffolds control mucilage architecture along with cellulosic rays and show that Arabidopsis SCE cells represent an excellent model in which to study the synthesis and function of GGM. Arabidopsis natural varieties with defects similar to muci10 mutants may reveal additional genes involved in GGM synthesis. Since GGM is the most abundant hemicellulose in the secondary walls of gymnosperms, understanding its biosynthesis may facilitate improvements in the production of valuable commodities from softwoods.

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine).

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.

Identification of genes involved in biosynthesis of mannan polysaccharides in Dendrobium officinale by RNA-seq analysis.

Dendrobium officinale is a traditional Chinese medicinal plant. The stems of D. officinale contain mannan polysaccharides, which are promising bioactive polysaccharides for use as drugs. However, the genes involved in the biosynthesis of mannan polysaccharides in D. officinale have not yet been identified. In this study, four digital gene expression profiling analyses were performed on developing stems of greenhouse-grown D. officinale to identify such genes. Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKE A genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database. In order to further analyze these DoCSLA genes, a full-length cDNA of each was obtained by RACE. The eight genes, belonging to the CSLA family of the CesA superfamily, contain conserved domains of the CesA superfamily. Most of the genes, which were highly expressed in the stems of D. officinale, were related to abiotic stress. Our results suggest that the CSLA family genes from D. officinale are involved in the biosynthesis of bioactive mannan polysaccharides.

CELLULOSE SYNTHASE-LIKE A2, a glucomannan synthase, is involved in maintaining adherent mucilage structure in Arabidopsis seed.

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-ß-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.

Identification of an additional protein involved in mannan biosynthesis.

Galactomannans comprise a ß-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it 'mannan synthesis-related' (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role.

Mannosylation in Candida albicans: role in cell wall function and immune recognition.

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post-translational modifications being the addition of O- and N-linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O- and N-linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host-fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.

Regulation of galactomannan biosynthesis in coffee seeds.

The seed of Coffea arabica accumulates large amounts of cell wall storage polysaccharides (CWSPs) of the mannan family in the cell walls of the endosperm. The variability induced by the growing environment and extensive pairwise correlation analysis with stringent significance thresholds was used to investigate transcript-transcript and transcript-metabolite relationships among 26 sugar-related genes, and the amount of CWSPs and seven soluble low molecular weight carbohydrates in the developing coffee endosperm. A dense module of nine quantitatively co-expressed genes was detected at the mid-developmental stage when CWSPs accumulate. This module included the five genes of the core galactomannan synthetic machinery, namely genes coding for the enzymes needed to assemble the mannan backbone (mannan synthase, ManS), and genes that introduce the galactosyl side chains (galactosyltransferase, GMGT), modulate the post-depositional degree of galactose substitution (α-galactosidase), and produce the nucleotide sugar building blocks GDP-mannose and UDP-galactose (mannose-1P guanyltransferase and UDP-glucose 4'-epimerase, respectively). The amount of CWSPs stored in the endosperm at the onset of their accumulation was primarily and quantitatively modulated at the transcriptional level (i.e. positively correlated with the expression level of these key galactomannan biosynthetic genes). This analysis also suggests a role for sorbitol and raffinose family oligosaccharides as transient auxiliary sources of building blocks for galactomannan synthesis. Finally, a microarray-based analysis of the developing seed transcriptome revealed that all genes of the core galactomannan synthesis machinery grouped in a single cluster of 209 co-expressed genes. Analysis of the gene composition of this cluster revealed remarkable functional coherence and identified transcription factors that putatively control galactomannan biosynthesis in coffee.

The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis.

We have proposed a new mannan catabolic pathway in Bacteroides fragilis NCTC 9343 that involves a putative mannanase ManA in glycoside hydrolase family 26 (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772). If this hypothesis is correct, ManA has to generate mannobiose from mannans as the major end product. In this study, the BF0771 gene from the B. fragilis genome was cloned and expressed in Escherichia coli cells. The expressed protein was found to produce mannobiose exclusively from mannans and initially from manno-oligosaccharides. Production of 4-O-ß-D-glucopyranosyl-D-mannose or 4-O-ß-D-mannopyranosyl-D-glucose from mannans was not detectable. The results indicate that this enzyme is a novel mannobiose-forming exo-mannanase, consistent with the new microbial mannan catabolic pathway we proposed.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Biosynthesis of the fungal cell wall polysaccharide galactomannan requires intraluminal GDP-mannose.

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy.

Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases.

The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1→3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA(O9a) has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two α-(1→3)- and two α-(1→2)-linked mannopyranose residues. In contrast, WbdA(O8) polymerizes trisaccharide repeat units containing single α-(1→3)-, α-(1→2)-, and ß-(1→2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.

Hemicellulose biosynthesis.

One major component of plant cell walls is a diverse group of polysaccharides, the hemicelluloses. Hemicelluloses constitute roughly one-third of the wall biomass and encompass the heteromannans, xyloglucan, heteroxylans, and mixed-linkage glucan. The fine structure of these polysaccharides, particularly their substitution, varies depending on the plant species and tissue type. The hemicelluloses are used in numerous industrial applications such as food additives as well as in medicinal applications. Their abundance in lignocellulosic feedstocks should not be overlooked, if the utilization of this renewable resource for fuels and other commodity chemicals becomes a reality. Fortunately, our understanding of the biosynthesis of the various hemicelluloses in the plant has increased enormously in recent years mainly through genetic approaches. Taking advantage of this knowledge has led to plant mutants with altered hemicellulosic structures demonstrating the importance of the hemicelluloses in plant growth and development. However, while we are on a solid trajectory in identifying all necessary genes/proteins involved in hemicellulose biosynthesis, future research is required to combine these single components and assemble them to gain a holistic mechanistic understanding of the biosynthesis of this important class of plant cell wall polysaccharides.