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Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described.

L-rhamnose isomerase (L-RhI) plays a key role in the microbial L-rhamnose metabolism by catalyzing the reversible isomerization of L-rhamnose to L-rhamnulose. Additionally, the enzyme exhibits activity on various other aldoses and ketoses, and its broad substrate specificity has attracted attention for its potential application in the production of rare sugars; however, improvement of the enzyme properties is desirable, such as thermal stability, enzymatic activity, and a pH optimum suitable for industrial usage. This review summarizes our current insights into L-RhIs with respect to their substrate recognition mechanism and their relationship with D-xylose isomerase (D-XI) based on structural and phylogenetic analyses. These two enzymes are inherently different, but recognize distinctly different substrates, and share common features that may be phylogenetically related. For example, they both have a flexible loop region that is involved in shaping active sites, and this region may also be responsible for various enzymatic properties of L-RhIs, such as substrate specificity and thermal stability. Key points • L-RhIs share structural features with D-XI. • There are two types of L-RhIs: E. coli L-RhI-type and D-XI-type. • Flexible loop regions are involved in the specific enzyme properties.

Rhamnolipids are a class of biosurfactants which contain rhamnose as the sugar moiety linked to β-hydroxylated fatty acid chains. Rhamnolipids can be widely applied in many industries including petroleum, food, agriculture and bioremediation etc. Pseudomonas aeruginosa is still the most competent producer of rhamnolipids, but its pathogenicity may cause safety and health concerns during large-scale production and applications. Therefore, extensive studies have been carried out to explore safe and economical methods to produce rhamnolipids. Various metabolic engineering efforts have also been applied to either P. aeruginosa for improving its rhamnolipid production and diminishing its pathogenicity, or to other non-pathogenic strains by introducing the key genes for safe production of rhamnolipids. The three key enzymes for rhamnolipid biosynthesis, RhlA, RhlB and RhlC, are found almost exclusively in Pseudomonas sp. and Burkholderia sp., but have been successfully expressed in several non-pathogenic host bacteria to produce rhamnolipids in large scales. The composition of mono- and di-rhamnolipids can also be modified through altering the expression levels of RhlB and RhlC. In addition, cell-free rhamnolipid synthesis by using the key enzymes and precursors from non-pathogenic sources is thought to not only eliminate pathogenic effects and simplify the downstream purification processes, but also to circumvent the complexity of quorum sensing system that regulates rhamnolipid biosynthesis. The pathogenicity of P. aeruginosa can also be reduced or eliminated through in vivo or in vitro enzymatic degradation of the toxins such as pyocyanin during rhamnolipid production. The rhamnolipid production cost can also be significantly reduced if rhamnolipid purification step can be bypassed, such as utilizing the fermentation broth or the rhamnolipid-producing strains directly in the industrial applications of rhamnolipids.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but little is known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release of L-rhamnose from the pectin substructure rhamnogalacturonan I, as well as catabolism of this monosaccharide. The rhaR disruptant was unable to grow on L-rhamnose, but only a small reduction in growth on pectin was observed. This is likely caused by the presence of a second, so far unknown regulator that responds to the presence of D-galacturonic acid.

Marine macroalgae, particularly their complex polysaccharides, are an untapped renewable source of high-quality monosaccharides and related building blocks. To utilize this feedstock for industrial applications, the enzymatic depolymerization by marine microorganisms has been shown to be effective. A prime example is the common green alga Ulva , with its storage polysaccharide ulvan, which contains high quantities of L -rhamnose and D -glucuronic acid. As suitable high-throughput methods for analyzing the enzymatic degradation of complex polysaccharides are still lacking, a transcription factor–based biosensor is described here that utilizes the P rha BAD promoter native to E. coli , which is specific for L -rhamnose. This biosensor exhibited a linear response, enabling the quantification of L -rhamnose within a concentration range of 10–1000 µM. The introduction of a T7 stem-loop improved the performance, and various fluorescent reporter genes were studied. The optimized system was then used to evaluate various stages of the ulvan degradation cascade in terms of L -rhamnose release, confirming its applicability to complex sugar mixtures. A detectable fluorescence signal was only generated when all the necessary enzymes for breaking down the polymer into undecorated monosaccharides were present, highlighting the biosensor’s specificity. The application of this method to the degradation of Ulva sp. biomass samples of various origins was also successfully demonstrated. This establishes the biosensor as a promising method for further high-throughput investigations. Key points •  Development of an improved transcription factor-based biosensor for L-rhamnose. •  Biosensor application for the analysis of enzymatic polysaccharide degradation. •  Reliable quantification of L-rhamnose in complex carbohydrate mixtures. Graphical Abstract

The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl -genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P . aeruginosa . Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P . putida and the native host P . aeruginosa . Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.

A novel l -rhamnose isomerase was identified and cloned from an extreme-temperature aquatic habitat metagenome. The deduced amino acid sequence homology suggested the possible source of this metagenomic sequence to be Chloroflexus islandicus . The gene expression was performed in a heterologous host, Escherichia coli , and the recombinant protein l -rhamnose isomerase (L-RI M ) was extracted and purified. The catalytic function of L-RI M was characterized for d -allulose to d -allose bioconversion. d -Allose is a sweet, rare sugar molecule with anti-tumour, anti-hypertensive, cryoprotective, and antioxidative properties. The characterization experiments showed L-RI M to be a Co ++ - or Mn ++ -dependent metalloenzyme. L-RI M was remarkably active (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RI M activity with d -allulose as the substrate occurred at pH 7.0 and 75 °C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively. L -RI M catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L −1 from 100 g L −1 d -allulose in 3 h. Key points • The present study explored an extreme temperature metagenome to identify a novel gene that encodes a thermostable l-rhamnose isomerase (L-RI M ) • L-RI M exhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges • L-RI M is excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively

In this study, a new l-rhamnose-binding lectin (GYL-R) from the hemolymph of bivalve Glycymeris yessoensis was purified using affinity and ion-exchange chromatography and functionally characterized. Lectin antimicrobial activity was examined in different ways. The lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, d-galactose, lactose, l-arabinose and raffinose. Using the glycan microarray approach, natural carbohydrate ligands were established for GYL-R as l-Rha and glycans containing the α-Gal residue in the terminal position. The GYL-R molecular mass determined by MALDI-TOF mass spectrometry was 30,415 Da. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 75 °C and between pH 4.0 and 12.0. The amino acid sequence of the five GYL-R segments was obtained with nano-ESI MS/MS and contained both YGR and DPC-peptide motifs which are conserved in most of the l-rhamnose-binding lectin carbohydrate recognition domains. Circular dichroism confirmed that GYL is a α/β-protein with a predominance of the random coil. Furthermore, GYL-R was able to bind and suppress the growth of the Gram-negative bacteria E. coli by recognizing lipopolysaccharides. Together, these results suggest that GYL-R is a new member of the RBL family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity.

A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 ( L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. Key points • Crystal structures of LrL-RhI in complexes with substrates were determined. • LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. • The LrL-RhI is active in acidic conditions.