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Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces , which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator ( rhaO ). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2 , by assembling the repressor/operator pair RhaR/ rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces . Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Methylotrophic yeast Ogataea polymorpha has become a promising cell factory due to its efficient utilization of methanol to produce high value-added chemicals. However, the low homologous recombination (HR) efficiency in O. polymorpha greatly hinders extensive metabolic engineering for industrial applications. Overexpression of HR-related genes successfully improved HR efficiency, which however brought cellular stress and reduced chemical production due to constitutive expression of the HR-related gene. Here, we engineered an HR repair pathway using the dynamically regulated gene ScRAD51 under the control of the l-rhamnose-induced promoter P LRA3 based on the previously constructed CRISPR-Cas9 system in O. polymorpha . Under the optimal inducible conditions, the appropriate expression level of ScRAD51 achieved up to 60% of HR rates without any detectable influence on cell growth in methanol, which was 10-fold higher than that of the wild-type strain. While adopting as the chassis strain for bioproductions, the dynamically regulated recombination system had 50% higher titers of fatty alcohols than that static regulation system. Therefore, this study provided a feasible platform in O. polymorpha for convenient genetic manipulation without perturbing cellular fitness.

2,4-Diketo-3-deoxy-l-rhamnonate (L-DKDR) hydrolase (LRA6) catalyzes the hydrolysis reaction of L-DKDR to pyruvate and l-lactate in the nonphosphorylated l-rhamnose pathway from bacteria and belongs to the fumarylacetoacetate hydrolase (FAH) superfamily. Most of the members of the FAH superfamily are involved in the microbial degradation of aromatic substances and share low sequence similarities with LRA6, by which the underlying catalytic mechanism remains unknown at the atomic level. We herein elucidated for the first time the crystal structures of LRA6 from Sphingomonas sp. without a ligand and in complex with pyruvate, in which a magnesium ion was coordinated with three acidic residues in the catalytic center. Structural, biochemical, and phylogenetic analyses suggested that LRA6 is a close but distinct subfamily of the fumarylpyruvate hydrolase (FPH) subfamily, and amino acid residues at equivalent position to 84 in LRA6 are related to different substrate specificities between them (Leu84 and Arg86 in LRA6 and FPH, respectively). Structural transition induced upon the binding of pyruvate was observed within a lid-like region, by which a glutamate-histidine dyad that is critical for catalysis was arranged sufficiently close to the ligand. Among several hydroxylpyruvates (2,4-diketo-5-hydroxycarboxylates), L-DKDR with a C6 methyl group was the best substrate for LRA6, conforming to the physiological role. Significant activity was also detected in acylpyruvate including acetylpyruvate. The structural analysis presented herein provides a more detailed understanding of the molecular evolution and physiological role of the FAH superfamily enzymes (e.g., the FAH like-enzyme involved in the mammalian l-fucose pathway).

Synthetic materials capable of engineering the immune system are of great relevance in the fight against cancer to replace or complement the current monoclonal antibody and cell therapy-based immunotherapeutics. Here, we report on antibody recruiting glycopolymers (ARGPs). ARGPs consist of polymeric copies of a rhamnose motif, which can bind endogenous antirhamnose antibodies present in human serum. As a proof-of-concept, we have designed ARGPs with a lipophilic end group that efficiently inserts into cell-surface membranes. We validate the specificity of rhamnose to attract antibodies from human serum to the target cell surface and demonstrate that ARGPs outperform an analogous small-molecule compound containing only one single rhamnose motif. The ARGP concept opens new avenues for the design of potent immunotherapeutics that mark target cells for destruction by the immune system through antibody-mediated effector functions.

d-Allose, a rare sugar, is an ideal table-sugar substitute and has many advantageous physiological functions. l-Rhamnose isomerase (l-RI) is an important d-allose-producing enzyme, but it exhibits comparatively low catalytic activity on d-allulose. In this study, an array of hydrophobic residues located within β1-α1-loop were solely or collectively replaced with polar amino acids by site-directed mutagenesis. A group of mutants was designed to weaken the hydrophobic environment and strengthen the catalytic behavior on d-allulose. Compared with that of the wild-type enzyme, the relative activities of the V48N/G59N/I63N and V48N/G59N/I63N/F335S mutants toward d-allulose were increased by 105.6 and 134.1%, respectively. Another group of mutants was designed to enhance thermostability. Finally, the t 1/2 values of mutant S81A were increased by 7.7 and 1.1 h at 70 and 80 °C, respectively. These results revealed that site-directed mutagenesis is efficient for improving thermostability and catalytic behavior toward d-allulose.

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli l-rhamnose P BAD promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an l-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an l-rhamnose catabolism deficient mutant, we demonstrate that the l-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to l-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated l-rhamnose transport and l-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an l-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an l-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1 H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1 H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, albeit with weak immunogenicity. In this study, the xenoantigen L-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via the anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity toward A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancers of tumor associated antigens in the development of tumor immunotherapies.

Advanced nanostructured materials, such as gold nanoparticles, magnetic nanoparticles, and multifunctional materials, are nowadays used in many state-of-the-art biomedical application. However, although the engineering in this field is very advanced, there remain some fundamental problems involving the interaction mechanisms between nanostructures and cells or tissues. Here we show the potential of (1)H NMR in the investigation of the uptake of two different kinds of nanostructures, that is, maghemite and gold nanoparticles, and of a chemotherapy drug (Temozolomide) in glioblastoma tumor cells. The proposed experimental protocol provides a new way to investigate the general problem of cellular uptake for a variety of biocompatible nanostructures and drugs.

The ability to profile the prevalence and functional activity of endogenous antibodies is of vast clinical and diagnostic importance. Serum antibodies are an important class of biomarkers and are also crucial elements of immune responses elicited by natural disease-causing agents as well as vaccines. In particular, materials for manipulating and/or enhancing immune responses toward disease-causing cells or viruses have exhibited significant promise for therapeutic applications. Antibody-recruiting molecules (ARMs), bifunctional organic molecules that redirect endogenous antibodies to pathological targets, thereby increasing their recognition and clearance by the immune system, have proven particularly interesting. Notably, although ARMs capable of hijacking antibodies against oligosaccharides and electron-poor aromatics have proven efficacious, systematic comparisons of the prevalence and effectiveness of natural anti-hapten antibody populations have not appeared in the literature. Herein we report head-to-head comparisons of three chemically simple antigens, which are known ligands for endogenous antibodies. Thus, we have chemically synthesized bifunctional molecules containing 2,4-dinitrophenyl (DNP), phosphorylcholine (PC), and rhamnose. We have then used a combination of ELISA, flow cytometry, and cell-viability assays to compare these antigens in terms of their abilities both to recruit natural antibody from human serum and also to direct serum-dependent cytotoxicity against target cells. These studies have revealed rhamnose to be the most efficacious of the synthetic antigens examined. Furthermore, analysis of 122 individual serum samples has afforded comprehensive insights into population-wide prevalence and isotype distributions of distinct anti-hapten antibody populations. In addition to providing a general platform for comparing and studying anti-hapten antibodies, these studies serve as a useful starting point for the optimization of antibody-recruiting molecules and other synthetic strategies for modulating human immunity.

Gold nanoparticles (AuNPs) are attractive materials for the immobilization of enzymes due to several advantages such as high enzyme loading, absence of internal diffusion limitations, and Brownian motion in solution, compared to the conventional immobilization onto porous macroscopic supports. The affinity of AuNPs to different groups present at the protein surface enables direct enzyme binding to the nanoparticle without the need of any coupling agent. Enzyme activity and stability appear to be improved when the biocatalyst is immobilized onto AuNPs. Rhamnulose-1-phosphate aldolase (RhuA) was selected as model enzyme for the immobilization onto AuNPs. The enzyme loading was characterized by four different techniques: surface plasmon resonance (SPR) shift and intensity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). AuNPs-RhuA complexes were further applied as biocatalyst of the aldol addition reaction between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal during two reaction cycles. In these conditions, an improved reaction yield and selectivity, together with a fourfold activity enhancement were observed, as compared to soluble RhuA., (© 2012 American Chemical Society)

In this study, a rhamnose-binding lectin from the roe of chinook salmon (Oncorhynchus tshawytscha) was purified and characterized, and its biological activities were examined in several model systems. Chinook salmon roe lectin had a molecular mass of 30 kDa and agglutinated rabbit and bovine erythrocytes. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 70 °C and between pH 4 and pH 11. Chinook salmon roe lectin did not exert antifungal activity toward the fungal species tested and did not exhibit mitogenic response toward mouse splenocytes up to a concentration of 5 mg/mL. The lectin had selective antiproliferative activity toward human breast cancer MCF-7 cells and hepatoma Hep G2 cells. It also induced the production of nitric oxide from mouse peritoneal macrophages. This is the first report that demonstrates these biological activities from chinook salmon roe lectin.

The targeting of autologous vaccines toward antigen presenting cells (APCs) via the in vivo complexation between anti α-Gal (anti-Gal) antibodies and α-Gal antigens presents a promising cancer immunotherapy with enhanced immunogenicity. This strategy takes advantage of the ubiquitous anti-Gal antibody in human serum. In contrast to the α-Gal epitope, the recent identification of high titers of anti-l-rhamnose (anti-Rha) antibodies in humans reveals a new approach toward immunotherapy employing l-rhamnose (Rha) monosaccharides. In order to evaluate this simple antigen in preclinical applications, we have synthesized Rha-conjugated immunogens and successfully induced high titers of anti-Rha antibodies in wildtype mice. Moreover, our studies demonstrate for the first time that wildtype mice could replace α1,3galactosyltransferase knockout (α1,3GT KO) mice in such antigen/antibody-mediated vaccine design when developing cancer immunotherapies.

Lipopolysaccharides (LPS) were isolated by hot phenol-water extraction from Danish Helicobacter pylori strains D1, D3, and D6, which were nontypeable using a variety of anti-Lewis and anti-blood-group monoclonal antibodies. An atypical O-chain polysaccharide (PS) was liberated from the LPS of the three strains by acid under mild conditions and found to contain D-rhamnose (D-Rha), L-rhamnose (L-Rha), and a branched sugar, 3-C-methyl-D-mannose (D-Man3CMe). The last sugar, which has not hitherto been found in Nature, was identified using GLC-MS of the derived alditol acetate and the partially methylated alditol acetate, and (1)H and (13)C NMR spectroscopy, including NOESY and (1)H,(13)C HMBC experiments. The following structure of the trisaccharide repeating unit of the PS was established: -->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D- Rhap-(1-- >. In contrast to the pathogenic importance of the Lewis antigen mimicry exhibited by the PS of H. pylori strains previously investigated, the biological relevance of the atypical PS for H. pylori pathogenesis is unclear. The production of a differing surface PS may represent a form of antigenic variation by these particular H. pylori strains and/or may reflect the adaptation of these strains to a particular human population.

The preparation of 17beta-substituted 14beta-hydroxysteroid C-3 alpha-L-rhamnopyranosides is described. These derivatives have a 14beta,20-ether, 14beta,20-lactone, or 17beta-CH2CH2OH, -CH2CH2NH2, -CH=CHNO2(E), -CH=CHCOOH(E), -CH(OH)CH2NO2(R), -CH(OMe)CH2NO2(R), -CH2-CH2COOH, or -CH(OH)CH2NH2(R) group. Derivatives were assayed in a radioligand binding assay for [3H]ouabain in membranes from canine heart muscle. The digitalis "receptor" comprises isoenzymes of the ion-pumping enzyme, Na+,K+-ATPase. The 17beta-CH=CHNO2(E), 17beta-CH=CHCOOH(E), and 17beta-CH(OMe)CH2NO2(R) derivatives were the most potent and equivalent to ouabain with low-nanomolar IC50 values. The very potent binding affinity of the disubstituted compound 17beta-CH(OMe)CH2NO2(R) further demonstrates that 17beta-unsaturated substitution is not required for potent binding affinity. This observation may be of value in the separation of cardiotonic and cardiotoxic effects. Tosylation of the 17beta-CH2OH, prepared from the 17beta-CHO by lithium aluminum hydride reduction, yielded the 14beta,17beta-ether. Synthesis of the 17beta-CH2COOH gave the epimeric 14alpha,17alpha- and 14beta,17beta-lactones. Structures have been established by NMR analysis.