Sugar-phosphate Research Articles

Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters.

Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by‐product are the main obstacles associated with the use of pyrophosphate in the phosphatase‐catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl‐compounds. Among them, acetyl phosphate delivered stable product levels and high phospho‐transfer efficiency at the lower functional pH‐limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative‐scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L−1 h−1 space‐time yield and 75% reduction of barium phosphate waste.

Comprehensive assessment of measurement uncertainty in 13C-based metabolic flux experiments

In the field of metabolic engineering 13C-based metabolic flux analysis experiments have proven successful in indicating points of action. As every step of this approach is affected by an inherent error, the aim of the present work is the comprehensive evaluation of factors contributing to the uncertainty of nonnaturally distributed C-isotopologue abundances as well as to the absolute flux value calculation. For this purpose, a previously published data set, analyzed in the course of a 13C labeling experiment studying glycolysis and the pentose phosphate pathway in a yeast cell factory, was used. Here, for isotopologue pattern analysis of these highly polar metabolites that occur in multiple isomeric forms, a gas chromatographic separation approach with preceding derivatization was used. This rendered a natural isotope interference correction step essential. Uncertainty estimation of the resulting C-isotopologue distribution was performed according to the EURACHEM guidelines with Monte Carlo simulation. It revealed a significant increase for low-abundance isotopologue fractions after application of the necessary correction step. For absolute flux value estimation, isotopologue fractions of various sugar phosphates, together with the assessed uncertainties, were used in a metabolic model describing the upper part of the central carbon metabolism. The findings pinpointed the influence of small isotopologue fractions as sources of error and highlight the need for improved model curation.Graphical abstractᅟ

A Description of Enzymatic Catalysis in N-Acetylhexosamine 1-Kinase: Concerted Mechanism of Two-Magnesium-Ion-Assisted GlcNAc Phosphorylation, Flexibility Behavior of Lid Motif upon Substrate Recognition, and Water-Assisted GlcNAc-1-P Release

The N-acetylhexosamine 1-kinase (NahK) is the typical example of anomeric kinases acting on gluco-type substrate, which catalyzes the phosphorylation of GlcNAc or GalNAc at anomeric C1 position with ATP, playing a crucial role in bifidobacteria metabolic pathway and biosynthesis of sugar 1-phosphates and oligosaccharides. Herein, by using state-of-the art ab initio QM/MM MD and MM MD simulations, one-dimensional and two-dimensional free energy profiles to descript catalytic process have been explored. A concerted mechanism has been recognized for the delivery of phosphate group and proton to product ADP and GlcNAc-1-P with the free energy barrier of ∼7.0 kcal/mol. The chemical reaction is assisted by two Mg2+ ions with a standing six coordination structure during the enzymatic process. The deficiency of Mg2+ bridging β–γ phosphates of ATP may alter the catalytic mechanism and brings higher free energy barriers. The protonation of GlcNAc-1-P is beneficial because its eliminates the Mg2+ ion binding. The wa...

Gas Chromatography/Mass Spectrometry-Based Metabolite Profiling of Nutrients and Antinutrients in Eight Lens and Lupinus Seeds (Fabaceae).

Lens culinaris and several Lupinus species are two legumes regarded as potential protein resources aside from their richness in phytochemicals. Consequently, characterization of their metabolite composition seems warranted to be considered as a sustainable commercial functional food. This study presents a discriminatory holistic approach for metabolite profiling in accessions of four lentil cultivars and four Lupinus species via gas chromatography/mass spectrometry. A total of 107 metabolites were identified, encompassing organic and amino acids, sugars, and sterols, along with antinutrients, viz., alkaloids and sugar phosphates. Among the examined specimens, four nutritionally valuable accessions ought to be prioritized for future breeding to include Lupinus hispanicus, enriched in organic ( ca. 11.7%) and amino acids ( ca. 5%), and Lupinus angustifolius, rich in sucrose ( ca. 40%), along with two dark-colored lentil cultivars 'verte du Puy' and 'Black Beluga' enriched in peptides. Antinutrient chemicals were observed in Lupinus polyphyllus, owing to its high alkaloid content. Several species-specific markers were also revealed using multivariate data analyses.

Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina.

We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-13C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze-thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves ( R2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-13C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation.

Trehalose 6-Phosphate Regulates Photosynthesis and Assimilate Partitioning in Reproductive Tissue.

Transgenic maize (Zea mays) that expresses rice (Oryza sativa) TREHALOSE PHOSPHATE PHOSPHATASE1 (TPP1) from the rice MADS6 promoter, which is active over the flowering period, produces higher yields than wild type. This yield increase occurs with or without drought conditions during flowering. To understand the mechanistic basis of the increased yield, we characterized gene expression and metabolite profiles in leaves and developing female reproductive tissue, comprising florets, node, pith, and shank, over the flowering period with and without drought. The MADS6 promoter was most active in the vasculature, particularly phloem companion cells in florets and pith, consistent with the largest decreases in trehalose 6-phosphate (T6P) levels (2- to 3-fold) being found in pith and florets. Low T6P led to decreased gene expression for primary metabolism and increased gene expression for secondary metabolism, particularly lipid-related pathways. Despite similar changes in gene expression, the pith and floret displayed opposing assimilate profiles: sugars, sugar phosphates, amino acids, and lipids increased in florets, but decreased in pith. Possibly explaining this assimilate distribution, seven SWEET genes were found to be up-regulated in the transgenic plants. SnRK1 activity and the expression of the gene for the SnRK1 beta subunit, expression of SnRK1 marker genes, and endogenous trehalose pathway genes were also altered. Furthermore, leaves of the transgenic maize maintained a higher photosynthetic rate for a longer period compared to wild type. In conclusion, we found that decreasing T6P in reproductive tissues down-regulates primary metabolism and up-regulates secondary metabolism, resulting in different metabolite profiles in component tissues. Our data implicate T6P/ SnRK1 as a major regulator of whole-plant resource allocation for crop yield improvement.

Improved phosphometabolome profiling applying isotope dilution strategy and capillary ion chromatography-tandem mass spectrometry

The phosphometabolome is comprised of all phosphorylated metabolites including the major metabolite classes sugar phosphates and nucleoside phosphates. Phosphometabolites are invaluable in any cell as a part of primary- and energy- metabolism, and as building blocks in the biosynthesis of macromolecules. Here, we report quantitative profiling of the phosphometabolome by applying capillary ion chromatography-tandem mass spectrometry (capIC-MS/MS), ensuring improved chromatographic separation, robustness and quantitative precision. Baseline separation was achieved for six out of eight tested hexose phosphates. Quantitative precision and reproducibility was improved by introducing a fully uniformly (U) 13C–labeled biological extract and applying an isotope dilution (ID) correction strategy. A 13C–labeled biological extract does in principle contain internal standards (IS) for all metabolites, but low abundant metabolites pose a challenge, and solutions to this are discussed. The extreme reproducibility and reliability of this capIC-MS/MS method was demonstrated by running the instrumentation continuously for ten days.

Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149

Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. β-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37–91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a β-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.

Abiotic Fabrication of Sugar Phosphates and Ribonucleosides in Water Microdroplets

Abiotic Fabrication of Sugar Phosphates and Ribonucleosides in Water Microdroplets

Sheathless Capillary Electrophoresis-Mass Spectrometry for theProfiling of Charged Metabolites in Biological Samples.

Capillary electrophoresis (CE) is well suited for the profiling of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. The protocol presented here is based on using a recently developed sheathless interfacing design, i.e., a porous tip interface, for coupling CE to electrospray ionization mass spectrometry (MS). It is demonstrated that sheathless CE-MS employing a bare fused-silica capillary at low-pH separation conditions can be used for the profiling of both cationic and anionic metabolites by only switching the MS detection and electrophoretic separation voltage polarity. The proposed sheathless CE-MS protocol allows efficient and sensitive profiles to be obtainedfor a broad array of charged metabolites, including amino acids, organic acids, nucleotides, and sugar phosphates, in various biological samples, such as urine and extracts of the glioblastoma cell line.

Quantification of Sugars and Organic Acids in Biological Matrices Using GC-QqQ-MS.

Gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ-MS) can be used to accurately quantify endogenous small molecules extracted from biological samples such as plants and human fluids including sera and urine. In order to quantify primary metabolites typically from central carbon metabolism such as sugars from glycolysis and the pentose phosphate pathway; and organic acids involved in the tricarboxylic acid (TCA) cycle; polar endogenous metabolites must be extracted from the samples of interest, chemically derivatized and quantified against a linear calibration curve to a corresponding authentic standard. This chapter describes how to quantify a combination of 48 primary metabolites belonging to classes of sugars, sugar alcohols, sugar acids, sugar phosphates, and organic acids using a robust, optimized, multiple reaction monitoring (MRM)-based GC-QqQ-MS method.

Genetic Investigation of Polymorphic OGG1 and MUTYH Genes Towards Increased Susceptibility in Lung Adenocarcinoma and its Impact on Overall Survival of Lung Cancer Patients Treated with Platinum Based Chemotherapy.

Genes OGG1 and MUTYH are the two primary genes in Base excision repair pathway. OGG1 hydrolyzes the sugar phosphate backbone and remove the damaged base creating abasic site. MUTYH complements OGG1 as it particularly remove adenine mispaired with 8-oxo-G. Both OGG1 and MUTYH act as a check for the mis-incorporation of bases may be due to damages incurred on DNA. DNA isolation for 326 lung cancer cases and 330 controls was followed by genotyping making use of PCR-RFLP. Logistic regression was done to analyze the risk towards lung cancer. Patients were followed through telephonic conversation. Kaplan meier and Cox-regression were used for survival analysis. OGG1 presented a high risk towards lung cancer (CG: OR = 2.44, p = 0.0003; CG + GG: OR = 1.88, p = 0.0093). On the same lines adenocarcinoma for OGG1 were potent risk factors towards lung cancer (CG: OR = 4.72, p = 0.0002; CG + GG: OR = 3.63, p = 0.0018). Single allelic carriers for MUTYH gene imposed a high risk towards overall lung susceptibility and for all the three histology. Stratified analysis for chemotherapeutic drugs revealed administration of Cisplatin/Carboplatin + Pemtrexed for OGG1Ser 326 Cys showed a better survival (MST CG vs. CC: 9.1 vs. 0.56, p = <0.0001; HR =0.051, p = 0.0025). Whereas, MUTYH Gln324His showed a smaller survival for mutant genotype (CC) (MST CC vs. GG: 4.0 vs. 9.4, p = 0.05; HR = 1.75, p = 0.26). Single allelic carriers for both OGG1 and MUTYH were risk factors towards lung cancer. The risk was amplified on combining both OGG1 and MUTYH.

Stringent Response Regulators Contribute to Recovery from Glucose Phosphate Stress in Escherichia coli.

In enteric bacteria such as Escherichia coli, the transcription factor SgrR and the small RNA SgrS regulate the response to glucose phosphate stress, a metabolic dysfunction that results in growth inhibition and stems from the intracellular accumulation of sugar phosphates. SgrR activates the transcription of sgrS, and SgrS helps to rescue cells from stress in part by inhibiting the uptake of stressor sugar phosphates. While the regulatory targets of this stress response are well described, less is known about how the SgrR-SgrS response itself is regulated. To further characterize the regulation of the glucose phosphate stress response, we screened global regulator gene mutants for growth changes during glucose phosphate stress. We found that deleting dksA, which encodes a regulator of the stringent response to nutrient starvation, decreases growth under glucose phosphate stress conditions. The stringent response alarmone regulator ppGpp (synthesized by RelA and SpoT) also contributes to recovery from glucose phosphate stress: as with dksA, mutating relA and spoT worsens the growth defect of an sgrS mutant during stress, although the sgrS relA spoT mutant defect was only detectable under lower stress levels. In addition, mutating dksA or relA and spoT lowers sgrS expression (as measured with a P sgrS -lacZ fusion), suggesting that the observed growth defects may be due to decreased induction of the glucose phosphate stress response or related targets. This regulatory effect could occur through altered sgrR transcription, as dksA and relA spoT mutants also exhibit decreased expression of a P sgrR -lacZ fusion. Taken together, this work supports a role for stringent response regulators in aiding the recovery from glucose phosphate stress.IMPORTANCE Glucose phosphate stress leads to growth inhibition in bacteria such as Escherichia coli when certain sugar phosphates accumulate in the cell. The transcription factor SgrR and the small RNA SgrS alleviate this stress in part by preventing further sugar phosphate transport. While the regulatory mechanisms of this response have been characterized, the regulation of the SgrR-SgrS response itself is not as well understood. Here, we describe a role for stringent response regulators DksA and ppGpp in the response to glucose phosphate stress. sgrS dksA and sgrS relA spoT mutants exhibit growth defects under glucose phosphate stress conditions. These defects may be due to a decrease in stress response induction, as deleting dksA or relA and spoT also results in decreased expression of sgrS and sgrR This research presents one of the first regulatory effects on the glucose phosphate stress response outside SgrR and SgrS and depicts a novel connection between these two metabolic stress responses.

Preparation of isotope dilution mass spectrometry standards based on glucose pulse

Isotope Dilution Mass Spectrometry (IDMS) is the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key is getting the corresponding fully uniformly(U) ¹³C-labeled metabolites to be measured. The conventional procedure for getting fully U ¹³C-labeled metabolites is through batch cultivation, but intracellular metabolites concentrations by this method are generally low. By applying U ¹³C-labeled glucose pulse, combined with fast sampling and quenching, mixture of fully U ¹³C-labeled intracellular metabolites was successfully extracted with higher concentration from Pichia pastoris G/DSEL fed with fully U ¹³C-labeled glucose as only carbon source. Quantitative results from liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS) show that concentrations of organic acids, sugar phosphates, amino acids and nucleotides were 2-10 folds higher than those without glucose pulse. Therefore, the glucose pulse method can efficiently improve the usage of fully U ¹³C-labeled glucose converting to ¹³C-labeled metabolites, and achieve the detection of intracellular metabolites with lower concentrate than the instrument detection limit.

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection

BackgroundUp to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb’s vulnerability and metabolic adaptation have been carried out.ResultsA genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports.ConclusionsThis study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Deciphering cyanobacterial phenotypes for fast photoautotrophic growth via isotopically nonstationary metabolic flux analysis

BackgroundSynechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium characterized to date. Its genome was found to be 99.8% identical to S. elongatus 7942 yet it grows twice as fast. Current genome-to-phenome mapping is still poorly performed for non-model organisms. Even for species with identical genomes, cell phenotypes can be strikingly different. To understand Synechococcus 2973’s fast-growth phenotype and its metabolic features advantageous to photo-biorefineries, 13C isotopically nonstationary metabolic flux analysis (INST-MFA), biomass compositional analysis, gene knockouts, and metabolite profiling were performed on both strains under various growth conditions.ResultsThe Synechococcus 2973 flux maps show substantial carbon flow through the Calvin cycle, glycolysis, photorespiration and pyruvate kinase, but minimal flux through the malic enzyme and oxidative pentose phosphate pathways under high light/CO2 conditions. During fast growth, its pool sizes of key metabolites in central pathways were lower than suboptimal growth. Synechococcus 2973 demonstrated similar flux ratios to Synechococcus 7942 (under fast growth conditions), but exhibited greater carbon assimilation, higher NADPH concentrations, higher energy charge (relative ATP ratio over ADP and AMP), less accumulation of glycogen, and potentially metabolite channeling. Furthermore, Synechococcus 2973 has very limited flux through the TCA pathway with small pool sizes of acetyl-CoA/TCA intermediates under all growth conditions.ConclusionsThis study employed flux analysis to investigate phenotypic heterogeneity among two cyanobacterial strains with near-identical genome background. The flux/metabolite profiling, biomass composition analysis, and genetic modification results elucidate a highly effective metabolic topology for CO2 assimilatory and biosynthesis in Synechococcus 2973. Comparisons across multiple Synechococcus strains indicate faster metabolism is also driven by proportional increases in both photosynthesis and key central pathway fluxes. Moreover, the flux distribution in Synechococcus 2973 supports the use of its strong sugar phosphate pathways for optimal bio-productions. The integrated methodologies in this study can be applied for characterizing non-model microbial metabolism.

Evolution of Rubisco activase gene in plants.

Rubisco activase of plants evolved in a stepwise manner without losing its function to adapt to the major evolutionary events including endosymbiosis and land colonization. Rubisco activase is an essential enzyme for photosynthesis, which removes inhibitory sugar phosphates from the active sites of Rubisco, a process necessary for Rubisco activation and carbon fixation. The gene probably evolved in cyanobacteria as different species differ for its presence. However, the gene is present in all other plant species. At least a single gene copy was maintained throughout plant evolution; but various genome and gene duplication events, which occurred during plant evolution, increased its copy number in some species. The exons and exon-intron junctions of present day higher plant's Rca, which is conserved in most species seem to have evolved in charophytes. A unique tandem duplication of Rca gene occurred in a common grass ancestor, and the two genes evolved differently for gene structure, sequence, and expression pattern. At the protein level, starting with a primitive form in cyanobacteria, RCA of chlorophytes evolved by integrating chloroplast transit peptide (cTP), and N-terminal domains to the ATPase, Rubisco recognition and C-terminal domains. The redox regulated C-terminal extension (CTE) and the associated alternate splicing mechanism, which splices the RCA-α and RCA-β isoforms were probably gained from another gene in charophytes, conserved in most species except the members of Solanaceae family.

Abiotic production of sugar phosphates and uridine ribonucleoside in aqueous microdroplets

Phosphorylation is an essential chemical reaction for life. This reaction generates fundamental cell components, including building blocks for RNA and DNA, phospholipids for cell walls, and adenosine triphosphate (ATP) for energy storage. However, phosphorylation reactions are thermodynamically unfavorable in solution. Consequently, a long-standing question in prebiotic chemistry is how abiotic phosphorylation occurs in biological compounds. We find that the phosphorylation of various sugars to form sugar-1-phosphates can proceed spontaneously in aqueous microdroplets containing a simple mixture of sugars and phosphoric acid. The yield for d-ribose-1-phosphate reached over 6% at room temperature, giving a ΔG value of -1.1 kcal/mol, much lower than the +5.4 kcal/mol for the reaction in bulk solution. The temperature dependence of the product yield for the phosphorylation in microdroplets revealed a negative enthalpy change (ΔH = -0.9 kcal/mol) and a negligible change of entropy (ΔS = 0.0007 kcal/mol·K). Thus, the spontaneous phosphorylation reaction in microdroplets occurred by overcoming the entropic hurdle of the reaction encountered in bulk solution. Moreover, uridine, a pyrimidine ribonucleoside, is generated in aqueous microdroplets containing d-ribose, phosphoric acid, and uracil, which suggests the possibility that microdroplets could serve as a prebiotic synthetic pathway for ribonucleosides.

Synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms.

Genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). However, engineering microbial workhorses for biomanufacturing of commodity compounds remains challenging. First, microbes cannot afford burdens with both overexpression of multiple enzymes and metabolite drainage for product synthesis. Second, synthetic circuits and introduced heterologous pathways are not yet as "robust and reliable" as native pathways due to hosts' innate regulations, especially under suboptimal fermentation conditions. Third, engineered enzymes may lack channeling capabilities for cascade-like transport of metabolites to overcome diffusion barriers or to avoid intermediate toxicity in the cytoplasmic environment. Fourth, moving engineered hosts from laboratory to industry is unreliable because genetic mutations and non-genetic cell-to-cell variations impair the large-scale fermentation outcomes. Therefore, synthetic biology strains often have unsatisfactory industrial performance (titer/yield/productivity). To overcome these problems, many different species are being explored for their metabolic strengths that can be leveraged to synthesize specific compounds. Here, we provide examples of non-conventional and genetically amenable species for industrial manufacturing, including the following: Corynebacterium glutamicum for its TCA cycle-derived biosynthesis, Yarrowia lipolytica for its biosynthesis of fatty acids and carotenoids, cyanobacteria for photosynthetic production from its sugar phosphate pathways, and Rhodococcus for its ability to biotransform recalcitrant feedstock. Finally, we discuss emerging technologies (e.g., genome-to-phenome mapping, single cell methods, and knowledge engineering) that may facilitate the development of novel cell factories.

Structural feature of calf thymus deoxyribonucleic acid–ruthenium(III) interaction in aqueous solution by difference Fourier transformed infrared spectroscopy

ABSTRACTHere is the report on the interaction of ruthenium(III) species with DNA in aqueous solution at pH 7.42 by Fourier transformed infrared difference spectroscopy. Under the physiological pH and molar ratio [Ruthenium]/[DNA] = 1/80–1/20 direct binding to guanine-N7, adenine-N7, and surprising binding to exocyclic thymine-O2 was found. At low metal concentration no significant shift of the absorption bands was observed, only nonspecific electrostatic binding of ruthenium(III) with negatively charged phosphate groups occurred. The increase of ruthenium(III) concentration caused DNA double helix destabilization and direct binding of the metal cation to guanine-N7 and thymine-O2. At higher ruthenium(III) concentrations denaturation of the DNA helix is evident with no apparent binding of ruthenium(III) to adenine and cytosine. Helix opening allows migration of ruthenium(III) ions from phosphate to available nucleobases (guanine and thymine). No alteration of the sugar phosphate geometry was observed thus confirming that DNA remains in B conformation.