Linear, unbranched (1,3;1,4)-β-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-β-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-β- and (1,4)-β-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-β-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

There has been a long-standing bottleneck in the quantitative analysis of the frequencies of homoblock polyads beyond triads using 1H and 13C NMR for linear polysaccharides, primarily because monosaccharides within a long homoblock share similar chemical environments due to identical neighboring units, resulting in indistinct NMR peaks. In this study, through rigorous mathematical induction, inequality relations were established that enabled the calculation of frequency ranges of homoblock polyads from historically reported NMR-derived frequency values of diads and/or triads of alginates, chitosans, homogalacturonans, and galactomannans. The calculated homoblock frequency ranges were then applied to evaluate three chain growth statistical models, including the Bernoulli chain, first-order Markov chain, and second-order Markov chain, for predicting homoblock frequencies in these polysaccharides. Furthermore, based on the mathematically derived inequality relations, a novel 2D array was constructed, enabling the graphical visualization of homoblock features in polysaccharides. It was demonstrated, as a proof of concept, that the novel 2D array, along with a 1D code generated from it, could serve as an effective feature engineering tool for polymer classification using machine learning algorithms.

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

The exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis have recently been shown to include (1,3;1,4)-β-D-glucans. In the present study, we examined another Clostridia bacterium Clostridium ventriculi that has long been considered to contain abundant amounts of cellulose in its exopolysaccharides. We treated alcohol insoluble residues of C. ventriculi that include the exopolysaccharides with the enzyme lichenase that specifically hydrolyses (1,3;1,4)-β-D-glucans, and examined the oligosaccharides released. This showed the presence of (1,3;1,4)-β-D-glucans, which may have previously been mistaken for cellulose. Through genomic analysis, we identified the two family 2 glycosyltransferase genes CvGT2–1 and CvGT2–2 as possible genes encoding (1,3;1,4)-β-D-glucan synthases. Gain-of-function experiments in the yeast Saccharomyces cerevisiae demonstrated that both of these genes do indeed encode (1,3;1,4)-β-D-glucan synthases.

beta-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of beta-chitin-derived high-value enzymes and products.

Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms. We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes. Chitin deacetylase (CDA) genes from each genome (PtCDA and TpCDA) were overexpressed in P. tricornutum. We performed comparative analysis of their sequence structure, phylogeny, transcriptional profiles, localization and enzymatic activities. The chitin relevant proteins show complex subcellular compartmentation. PtCDA was likely acquired by horizontal gene transfer from prokaryotes, whereas TpCDA has closer relationships with sequences in Opisthokonta. Using transgenic P. tricornutum lines expressing CDA-green fluorescent protein (GFP) fusion proteins, PtCDA predominantly localizes to Golgi apparatus whereas TpCDA localizes to endoplasmic reticulum/chloroplast endoplasmic reticulum membrane. CDA-GFP overexpression upregulated the transcription of chitin synthases and potentially enhanced the ability of chitin synthesis. Although both CDAs are active on GlcNAc(5), TpCDA is more active on the highly acetylated chitin polymer DA60. We have addressed the ambiguous characters of CDAs from P. tricornutum and T. pseudonana. Differences in localization, evolution, expression and activities provide explanations underlying the greater potential of centric diatoms for chitin biosynthesis. This study paves the way for in vitro applications of novel CDAs.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

An oligosaccharide was isolated in high purity and excellent yield from the water-extractable mucilage of chia (Salvia hispanica L.) seeds using an optimized solid-phase extraction method. LC–MS analysis showed that the compound presents a molecular mass of 504 Da and trifluoroacetic acid hydrolysis revealed that it consists of galactose, glucose and fructose. Glycosidic linkage analysis showed that the oligosaccharide contains two non-reducing ends corresponding to terminal glucopyranose and terminal galactopyranose, respectively. The oligosaccharide was identified as planteose by the complete assignment of a series of 2D NMR spectra (COSY, TOCSY, ROESY, HSQC, and HMBC). The significance of the presence of planteose in chia seeds is discussed in the context of nutrition and food applications.

Mucilages are hydrocolloid solutions produced by plants for a variety of functions, including the creation of a water-holding barrier around seeds. Here we report our discovery of the formation of three distinct mucilage layers around Plantago ovata seeds upon their hydration. Each layer is dominated by different arabinoxylans (AXs). These AXs are unusual because they are highly branched and contain β-1,3-linked xylose in their side chains. We show that these AXs have similar monosaccharide and linkage composition, but vary in their polymer conformation. They also exhibit distinct rheological properties in aqueous solution, despite analytical techniques including NMR showing little difference between them. Using enzymatic hydrolysis and chaotropic solvents, we reveal that hydrogen bonding and side chain distribution are key factors underpinning the distinct rheological properties of these complex AXs.