Glycosyltransferase Research Articles

Complete genome sequence provides insights into the biodrying-related microbial function of Bacillus thermoamylovorans isolated from sewage sludge biodrying material

To enable the development of microbial agents and identify suitable candidate used for biodrying, the existence and function of Bacillus thermoamylovorans during sewage sludge biodrying merits investigation. This study isolated a strain of B. thermoamylovorans during sludge biodrying, submitted it for complete genome sequencing and analyzed its potential microbial functions. After biodrying, the moisture content of the biodrying material decreased from 66.33% to 50.18%, and B. thermoamylovorans was the ecologically dominant Bacillus, with the primary annotations associated with amino acid transport and metabolism (9.53%) and carbohydrate transport and metabolism (8.14%). It contains 96 carbohydrate-active- enzyme-encoding gene counts, mainly distributed in glycoside hydrolases (33.3%) and glycosyl transferases (27.1%). The virulence factors are mainly associated with biosynthesis of capsule and polysaccharide capsule. This work indicates that among the biodrying microorganisms, B. thermoamylovorans has good potential for degrading recalcitrant and readily degradable components, thus being a potential microbial agent used to improve biodrying.

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon.

Summary The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed.We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait.Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the Bd GT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the Bd GT43A gene in Brachypodium.The biomass of the silenced lines showed higher digestibility supporting a causative role of the Bd GT43A gene, suggesting that it might form a good target for improving straw digestibility in crops.

Proteins isolated of Pueraria Radix possible to cause allergenic react with immunoglobulin E in human sera

Allergies occur when immunoglobulin E (IgE), which is part of the body’s immune system, binds to molecules. A protein in the foods, plants or pollen is usually the problem. This causes the release of inflammatory chemicals such as histamine. There are a few cases where allergies are cause by proteins derived from herbal medicine, but we do not know yet what protein causes the immune react to IgE. Here, we demonstrate to confirm if the Pueraria Radix at the proteomic level triggers IgE immunoreactivity. Finally, we present the molecules of the Pueraria Radix-derived protein that trigger the IgE immune response. To prove our hypothesis, we used proteomic tools and SDS-PAGE stained with Coomassie blue and identified proteins using LC-MS/ MS. Proteins were separated from Pueraria Radix and allergic reaction sera were selected. We found three positive sera that showed an immune response to Pueraria Radix protein. Positive sera demonstrated a similar pattern of IgE reactivity and mostly restricted to a 50-70 kDa band. Therefore, we have identified number of different IgE binding proteins. Therefore, IgE binding proteins were identified such as enolase, hypothetical protein, DING protein and glycosyl transferase. The results imply that Pueraria Radix can act as an allergen given the identification of IgE binding proteins among the isolated proteins of Pueraria Radix.

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1- mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1- strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of the potential role of microorganisms from a metagenomic analysis

Sewage sludge biodrying is a waste treatment method that uses bio-heat generated from organic degradation to remove moisture from sewage sludge. Lignocellulose and carbohydrate decomposition is important when assessing biodrying performance. This study investigated lignocellulose and carbohydrate decomposition, and the potential microbial functions during biodrying. We determined the lignocellulose and carbohydrate contents, assayed related enzyme activity, performed a complete metagenomic study on sewage sludge biodrying material during the thermophilic phase, annotated potential genetic function involved in the decomposition, and summarized the key metabolic pathways. The results indicated that lignocellulose, readily degradable carbohydrates, and starch, significantly decomposed after biodrying. During the thermophilic phase, the majority of lignocellulose and carbohydrate-related enzymes showed significantly higher activity, and glycoside hydrolases and glycosyl transferases showed higher gene counts and reads. Moreover, the top five microorganisms enriched with carbohydrate-active enzyme genes, i.e., Bacillus, Intrasporangium, Tetrasphaera, Rhodobacter, and Streptomyces, were also among the top ten ecologically dominant genera. These findings highlight the crucial phases for biodrying process, reveal the ecologically functional diversity of biodrying-originated microbial consortia, and suggest potential candidates for optimizing biodrying decomposition.

Boronic-Acid-Catalyzed Regioselective and 1,2- cis-Stereoselective Glycosylation of Unprotected Sugar Acceptors via SNi-Type Mechanism.

Regio- and 1,2- cis-stereoselective chemical glycosylation of unprotected glycosyl acceptors has been in great demand for the efficient synthesis of natural glycosides. However, simultaneously regulating these selectivities has been a longstanding problem in synthetic organic chemistry. In nature, glycosyl transferases catalyze regioselective 1,2- cis-glycosylations via the SNi mechanism, yet no useful chemical glycosylations based on this mechanism have been developed. In this paper, we report a highly regio- and 1,2- cis-stereoselective SNi-type glycosylation of 1,2-anhydro donors and unprotected sugar acceptors using p-nitrophenylboronic acid (10e) as a catalyst in the presence of water under mild conditions. Highly controlled regio- and 1,2- cis-stereoselectivities were achieved via the combination of boron-mediated carbohydrate recognition and the SNi-type mechanism. Mechanistic studies using the KIEs and DFT calculations were consistent with a highly dissociative concerted SNi mechanism. This glycosylation method was applied successfully to the direct glycosylation of unprotected natural glycosides and the efficient synthesis of a complex oligosaccharide with minimal protecting groups.

Transcriptomic responses in Neolitsea sericea leaves under acute drought stress

Neolitsea sericea is acclimated to arid islands, but few studies have examined its responses to water stress. To look into the defending responses that assist N. sericea seedlings to survive drought, physiological and transcriptomic analysis of leaves was conducted after drought was induced. Over 17,768,244 reads for each sample were generated, totalling over 4400 million base pairs (bp). Among 129,239 unigenes that were assembly yielded with an average length of 816 bp, 51,137 of them (39.6%) were annotated successfully. Under severe drought, physiological inhibition was strengthened and abscisic acid content was significantly upregulated. The responses were strongly accompanied by transcriptional regulation of genes participating in stress perception, protective signalling, hormone metabolism, transcription factors, abiotic stress, transport, and degradation. In total, 61, 65, and 67 differentially expressed genes were identified after 12, 24, and 72 h exposure to severe drought stress, respectively. Gene Ontology and MapMan functional enrichment analysis revealed that the most upregulated genes were involved in protein transport, the regulation of transcription, and miscellaneous enzyme families. In addition, genes that encode glycosyl transferase and UDP-glycosyltransferases may modulate the drought-stress responses of N. sericea. Multiple hormones were active in N. sericea leaves during drought stress, including those associated with auxins, abscisic acid, brassinosteroid, and cytokinins. Transcription factors, including AtbHLH112, AtCOL4, AtZFP3, AtCIR1, and AtCCA1, may participate in ABA signal transduction in drought-treated N. sericea. The present study elucidates how N. sericea make transcriptomic responses to drought stress, helping to reveal the molecular mechanisms connected to drought adaptation.

High throughput mass spectrometry-based characterisation of Arabidopsis thaliana group H glycosyltransferases.

In this report, we cloned and characterised four members of group H glycosyltransferases (GTs) by studying their substrate specificities and kinetics. The formation of products and possible glycosylation position was confirmed using MS/MS. The results revealed that 76E1 and 76E5 have broader donor specificity, including UDP-glucose (UDPGlc), UDP-galactose (UDPGal) and UDP-N-acetylglucosamine (UDPGlcNAc) with various flavonoids as acceptor substrates. Pseudo-single substrate kinetics data showed a relatively low KM, indicating a high affinity for substrate UDPGlc and also supported that 76E5 is more of a galactosyl and N-acetylglucosamine transferase. Sequence alignment and site-directed mutagenesis studies indeed suggested that serine is a crucial residue in the UDPGlcNAc and UDPGal activity.

Overexpression of the <i>Rosa rugosa RrGT1</i> Gene Induces Anthocyanin Accumulation in Tobacco

Rosa rugosa has always been an important plant in landscape application, and the improvements and innovations about its flower color are particularly important. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, were isolated from flowers of R. rugosa “Zizhi” and then functionally characterized. Sequence alignments with the NCBI database show that the RrGT1 protein is a member of the GTB superfamily and has typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa “Zizhi” and their expression patterns corresponded with the accumulation of anthocyanins. Additionally, the in vivo function of RrGT1 was investigated via its overexpression in tobacco. Transgenic tobacco plants expressing RrGT1 induced anthocyanin accumulation in flowers, indicating that RrGT1 could encode a functional glycosyltransferase (GT) protein for anthocyanin biosynthesis and could function in other species. Therefore, we speculated that glycosylation of RrGT1 played a crucial role in anthocyanin biosynthesis in R. rugosa.

New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli

Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.

Osteoblastic Cell-Derived Extracellular Vesicles Transfer Small RNAs That Alter the Physiology of Hematopoietic Cells In Vivo

BackgroundThe bone marrow microenvironment (BMEV) regulates the highly regenerative hematopoietic system. However, there are a limited number of BMEV-derived molecules with a definitive role in maintaining hematopoietic stem and progenitor cells (HSPCs).Extracellular vesicles (EVs) encapsulate bioactive molecules, and may modify the physiology of their target cells. In hematopoiesis, EVs derived from culture-expanded mesenchymal cells can rescue irradiation damage, expand human umbilical cord blood cells and support HSPCs in vitro . However, in vivo evidence of EV function is lacking. We therefore sought to investigate the role of EVs in the interaction between the BMEV and the hematopoietic system and took advantage of existing mice bearing genetic reporters of key mesenchymal cell types.ResultsWhile analyzing the bone marrow (BM) of different mesenchymal cell-GFP reporter mice, we unexpectedly found CD45+ GFP+ cells. These were confirmed as single cells with intracellular GFP as demonstrated by imaging flow cytometry and confocal microscopy (Fig. 1A). Moreover, their hematopoietic identity was confirmed by their ability to form myeloid colonies in methylcellulose.Transplanted CD45.1 BM into Osteocalcin-Topaz (Ocn-Topaz) and Collagen1-GFP (Col1-GFP) mice that label osteoblasts, as well as Nestin-GFP (Nes-GFP) that labels mesenchymal stem cells demonstrated that donor cells are comparably labeled with GFP in Ocn-Topaz and Col1-GFP (2.2%) but at a much lower frequency (0.05%) in Nes-GFP. We therefore decided to proceed with the Ocn-Topaz model to investigate the role of osteoblast derived EVs in hematopoietic communication.Within the lineage negative compartment, the frequency of GFP+ cells increased with maturation. The highest frequency found in GMPs (0.06% of live cells were GFP+), followed by CMPs (0.01%), MEPs (0.002%) and LKS (0.004%) (Fig. 1B). Of particular interest, Lin- GFP+ cells formed ~5 fold more colonies as compared to their GFP- counterparts. However, transplantation assays demonstrated that the GFP+ cells possessed a decreased ability for long term reconstitution.Given the molecular weight of GFP, we hypothesized that EVs were the basis for transfer. Transmission electron microscopy coupled with immunogold staining revealed microvesicular structures of ~100 nm in size that contained GFP and that were labeled with the exosome marker TSG101 (Fig. 1C). Western blotting and flow cytometry detected labeling with exosome markers CD81 and CD9.Heparin sulfate proteoglycans (HSPGs) have been implicated in the biogenesis and uptake of EVs. Osteoblast-specific disruption of HSPGs by the knock out of the glycosyl transferase EXT1 resulted in a (40%) drop in the frequency of GFP+ cells in the GMP compartment. These findings demonstrate the EV-dependent transfer of GFP from osteoblasts to BM hematopoietic cells, and confirm GFP as a marker for the isolation and characterization of EV target cells.Exosomes from the BM of Ocn-Topaz mice in addition to GFP+ and GFP- GMPs were isolated for small RNA sequencing. In parallel, GMP populations were collected for mRNA sequencing. Global analysis of small RNA libraries from EVs and GMPs demonstrated that piRNAs was the most abundant species in both EVs (30%) and GMPs (18%). Surprisingly, EVs had low miRNA content (1.4%) compared to GMPs (9.2%) (Fig. 1D). When comparing GFP+ GMPs to GFP- ones, 6 miRNAs (mir-143, mir-122, mir-423-5p, mir-451, mir-206, mir-146b*) showed at least 100% increase in the GFP+GMPs. Predicted targets of mir-143, mir-206, mir-146 emerged as enriched sets when comparing gene expression of GFP+ and GFP- GMPs.In contrast, tRNAs was the most enriched species in EVs (10.5%) when compared to GMPs (2.5%) (Fig. 1D) and interestingly, GFP+ GMPs had higher content of tRNA when compared to GFP- (3.3% vs 1.7%) respectively. Given the role of tRNAs in translation and the emerging role of tRNA fragments (tRFs) in translation regulation and stress signaling, it was of interest to see translation and ribosome genesis among the top enriched gene sets when comparing GFP+ and GFP- GMPs.In conclusion, we present evidence for the in vivo transfer of bioactive EVs from osteoblasts to BM progenitor populations, and that this transfer alters hematopoietic cell function and gene expression. Moreover, we identify piRNAs and tRNAs as the most enriched species of small RNAs within BM derived EVs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of six glycosyltransferases involved in the biosynthesis of a new bacterial exopolysaccharide in Paenibacillus elgii.

Paenibacillus elgii B69 produces a new xylose-containing exopolysaccharide (EPS) that effectively removes the pollutants from wastewater through flocculation. However, information about the biosynthesis of this EPS is limited. In this study, sequence analysis showed six putative glycosyltransferases (GTs) genes in polysaccharide gene clusters involved in glycosidic linkages of repeating units. Each gene was deleted and phenotypes were examined to understand the functions of these genes. Two of the genes were deleted successfully to encode a priming glucose GT and a side-chain xylose GT, but other genes were unsuccessfully deleted because of the accumulation of toxic intermediate products. The six genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified. The activity of GTs was analyzed through mass spectrometry by using the purified membrane fraction as a lipid carrier receptor after a hexasaccharide repeated unit was reconstructed in vitro. The specificities of six different GTs and the building order of the hexasaccharide were characterized. This study provided a basis for future research on the biosynthetic pathway of EPS in Paenibacillus or other genera.

Cell Wall Associated Factors of Mycobacterium tuberculosis as Major Virulence Determinants: Current Perspectives in Drugs Discovery and Design.

Mycobacteria genus is responsible for deadly diseases like tuberculosis and leprosy. Cell wall of bacteria belonging to this genus is unique in many ways. It plays a major role in the pathogenesis and intracellular survival inside the host. In intracellular pathogens, their cell wall acts as molecular shield and interacts with host cell milieu to modulate host defense responses. In this review, we summarize the factors that participate in the biosynthesis of unique mycobacterial cell wall, understand their potential as drug targets and the recent developments where they have been evaluated as possible drug targets. Several cell wall associated factors that play crucial roles in the synthesis of cell wall components like Antigen 85 complex, Glycosyltransferases (GTs), LM (lipomannan) and LAM (lipoarabinomannan), mAGP Complex, lipolytic enzyme have been categorically documented. Most of the presently used anti TB regimens interrupted cell wall synthesis, but the emergence of drug resistant strains made it mandatory to identify new drug targets. Novel drug candidates which could inhibit the synthesis of cell wall components have been thoroughly studied worldwide. Studies demonstrated that the cell wall components are unique in terms of their contribution in mycobacterium pathogenesis. Targeting these can hamper the growth of M. tuberculosis. In this study, we scrutinize the drugs under trials and the potential candidates screened through in silico findings.

Screening, Partial Purification and Stability Studies of Cyclodextrin glycosyl transferases from Bacillus amyloliquefaciens Strain L4-6

Screening, Partial Purification and Stability Studies of Cyclodextrin glycosyl transferases from Bacillus amyloliquefaciens Strain L4-6

The Glycosyltransferases of LPS Core: A Review of Four Heptosyltransferase Enzymes in Context.

Bacterial antibiotic resistance is a rapidly expanding problem in the world today. Functionalization of the outer membrane of Gram-negative bacteria provides protection from extracellular antimicrobials, and serves as an innate resistance mechanism. Lipopolysaccharides (LPS) are a major cell-surface component of Gram-negative bacteria that contribute to protecting the bacterium from extracellular threats. LPS is biosynthesized by the sequential addition of sugar moieties by a number of glycosyltransferases (GTs). Heptosyltransferases catalyze the addition of multiple heptose sugars to form the core region of LPS; there are at most four heptosyltransferases found in all Gram-negative bacteria. The most studied of the four is HepI. Cells deficient in HepI display a truncated LPS on their cell surface, causing them to be more susceptible to hydrophobic antibiotics. HepI–IV are all structurally similar members of the GT-B structural family, a class of enzymes that have been found to be highly dynamic. Understanding conformational changes of heptosyltransferases are important to efficiently inhibiting them, but also contributing to the understanding of all GT-B enzymes. Finding new and smarter methods to inhibit bacterial growth is crucial, and the Heptosyltransferases may provide an important model for how to inhibit many GT-B enzymes.

Structural Snapshots of α-1,3-Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases.

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double-displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α-1,3-galactosyltransferase (α3GalT) from Bos taurus, which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP-Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front-side substrate-assisted SN i-type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.

The O-antigen structure of bacterium Comamonas aquatica CJG.

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1H and 13C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry

BackgroundTrichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass.ResultsIn this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins.ConclusionsIdentifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

Hybrid Sequencing of Full-Length cDNA Transcripts of Stems and Leaves in Dendrobium officinale

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem.

Elucidating the Sugar Tailoring Steps in the Cytorhodin Biosynthetic Pathway.

Anthracycline antitumor cytorhodins X and Y feature a rare 9α-glycoside and 7-dexoy-aglycone. Characterization of the cytorhodin gene cluster from Streptomyces sp. SCSIO 1666 through gene inactivations and metabolite analyses reveals three glycosyltransferases (GTs) involved in the sugar tailoring steps. The duo of CytG1 and CytL effects C-7 glycosylation with l-rhodosamine whereas the iterative GT CytG3 and CytW similarly modifies both C-9 and C-10 positions. CytG2 also acts iteratively by incorporating the second and third sugar moiety into the trisaccharide chains at the C-7 or C-10 position.