Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol.

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Using Native and Synthetic Genes to Disrupt Inositol Pyrophosphates and Phosphate Accumulation in Plants.

Inositol pyrophosphates are eukaryotic signaling molecules that have been recently identified as key regulators of plant phosphate sensing and homeostasis. Given the importance of phosphate to current and future agronomic practices, we sought to design plants which could be used to sequester phosphate, as a step in a phytoremediation strategy. To achieve this, we expressed Diadenosine and Diphosphoinositol Polyphosphate Phosphohydrolase (DDP1), a yeast (Saccharomyces cerevisiae) enzyme demonstrated to hydrolyze inositol pyrophosphates, in Arabidopsis thaliana and pennycress (Thlaspi arvense), a spring annual cover crop with emerging importance as a biofuel crop. DDP1 expression in Arabidopsis decreased inositol pyrophosphates, activated Phosphate Starvation Response marker genes, and increased phosphate accumulation. These changes corresponded with alterations in plant growth and sensitivity to exogenously applied phosphate. Pennycress plants expressing DDP1 displayed increases in phosphate accumulation, suggesting that these plants could potentially serve to reclaim phosphate from phosphate-polluted soils. We also identified a native Arabidopsis gene, Nucleoside diphosphate-linked moiety X 13 (NUDIX13), which we show encodes an enzyme homologous to DDP1 with similar substrate specificity. Arabidopsis transgenics overexpressing NUDIX13 had lower inositol pyrophosphate levels and displayed phenotypes similar to DDP1-overexpressing transgenics, while nudix13-1 mutants had increased levels of inositol pyrophosphates. Taken together, our data demonstrates that DDP1 and NUDIX13 can be used in strategies to regulate plant inositol pyrophosphates and could serve as potential targets for engineering plants to reclaim phosphate from polluted environments.

Novel treatment for PXE: Recombinant ENPP1 enzyme therapy.

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of a potent endogenous calcification inhibitor, plasma inorganic pyrophosphate (PPi); the deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1 that generates PPi and is now in clinical trials, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of Abcc6-/-mice. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6-/- mice. When Abcc6-/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 h. When Abcc6-/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising second generation enzyme therapy for PXE, the first generation of which is currently in clinical testing.

Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

IQGAP-2: A novel interacting partner for the Human Colonic Thiamin Pyrophosphate Transporter (hcTPPT).

The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B1 (i. e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affects its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library, and have identified 3 putative interactors, namely IQGAP-2, SNX-6 and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and co-immunoprecipitation approaches) the putative interactor to co-localize with hcTPPT, and to directly interact with the transporter. Also, over-expressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (P < 0.01) induction in TPP uptake, while it's knocking down (using gene-specific siRNAs) caused significant (P < 0.01 & < 0.05) decrease in uptake. Furthermore, over-expressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.

On-demand utilization of phosphoribosyl pyrophosphate by downstream anabolic pathways.

The pentose phosphate pathway (PPP) is critical for anabolism and biomass production. Here we show that the essential function of PPP in yeast is the synthesis of phosphoribosyl pyrophosphate (PRPP) catalyzed by PRPP-synthetase. Using combinations of yeast mutants, we found that a mildly decreased synthesis of PRPP affects biomass production, resulting in reduced cell size, while a more severe decrease ends up affecting yeast doubling time. We establish that it is PRPP itself that is limiting in invalid PRPP-synthetase mutants and that the resulting metabolic and growth defect can be bypassed by proper supplementation of the medium with ribose-containing precursors or by the expression of bacterial or human PRPP-synthetase. In addition, using documented pathologic human hyperactive forms of PRPP-synthetase, we show that intracellular PRPP as well as its derived products can be increased in both human and yeast cells, and we describe the ensuing metabolic and physiological consequences. Finally, we found that PRPP consumption appears to take place "on demand" by the various PRPP-utilizing pathways, as shown by blocking or increasing the flux in specific PRPP-consuming metabolic routes. Overall, our work reveals important similarities between human and yeast for both synthesis and consumption of PRPP.

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

The Property of a Key Amino Acid Determines the Function of Farnesyl Pyrophosphate Synthase in Sporobolomyces pararoseus NGR.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the synthesis of C15 farnesyl diphosphate (FPP) from C5 dimethylallyl diphosphate (DMAPP) and two or three C5 isopentenyl diphosphates (IPPs). FPP is an important precursor for the synthesis of isoprenoids and is involved in multiple metabolic pathways. Here, farnesyl pyrophosphate synthase from Sporobolomyces pararoseus NGR (SpFPPS) was isolated and expressed by the prokaryotic expression system. The SpFPPS full-length genomic DNA and cDNA are 1566 bp and 1053 bp, respectively. This gene encodes a 350-amino acid protein with a predicted molecular mass of 40.33 kDa and a molecular weight of 58.03 kDa (40.33 kDa + 17.7 kDa), as detected by SDS-PAGE. The function of SpFPPS was identified by induction, purification, protein concentration and in vitro enzymatic activity experiments. Structural analysis showed that Y90 was essential for chain termination and changing the substrate scope. Site-directed mutation of Y90 to the smaller side-chain amino acids alanine (A) and lysine (K) showed in vitro that wt-SpFPPS catalyzed the condensation of the substrate DMAPP or geranyl diphosphate (GPP) with IPP at apparent saturation to synthesize FPP as the sole product and that the mutant protein SpFPPS-Y90A synthesized FPP and C20 geranylgeranyl diphosphate (GGPP), while SpFPPS-Y90K hydrolyzed the substrate GGPP. Our results showed that FPPS in S. pararoseus encodes the SpFPPS protein and that the amino acid substitution at Y90 changed the distribution of SpFPPS-catalyzed products. This provides a baseline for potentially regulating SpFPPS downstream products and improving the carotenoid biosynthesis pathway.

Effect of knocking out mouse Slc44a4 on colonic uptake of the microbiota-generated thiamine pyrophosphate and colon physiology.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.

Enhanced pyrophosphate detection: Utilizing oPD-derived carbon dots and Fe3+ interactions in a paper strip biosensor.

The development of portable, cost-effective, and straightforward DNA biosensors holds immense importance in various fields, including healthcare, environmental monitoring, and food safety. This study contributes to the objective by introducing an innovative approach for synthesizing carbon dots (Cdots) with high quantum yield (QY) and remarkable selectivity for Fe3+ ions. Utilizing o-phenylenediamine as a precursor, the study achieved a straightforward and environmentally friendly synthesis method, enabling the efficient detachment of metal ions from the Cdot surface upon introducing pyrophosphate (PPi). The presence of surface hydroxyl and amino groups facilitated specific Fe3+ recognition. Employing D-optimal response surface methodology, the study optimized Cdot synthesis parameters, identifying temperature and heating time as critical factors influencing QY. Statistical analysis confirmed the model's reliability, predicting maximum QY of 48.8 % with minimal deviation from experimental results. Characterization studies revealed the amorphous nature of Cdots through HR-TEM, XRD, and FTIR analysis. Furthermore, the proposed LAMP/PPi biosensing technique demonstrated higher sensitivity, specificity, and repeatability, with negligible interference from common anions and efficacy across varying pH levels. The limit of detection (LOD) of 0.079 (±0.01) µM and the detection range of 0.1 µM-2 mM underscore the biosensor's practical utility. This study highlights a promising direction for developing paper-based LAMP/PPi biosensors with potential diagnostics and environmental monitoring applications. Significantly, the biosensing technique is applicable to any DNA amplification method generating pyrophosphate (PPi) as a by-product.

Significant Enhancement of Negative Thermal Expansion Under Low Pressure in Cu2P2O7.

Much effort is made to achieve the negative thermal expansion (NTE) control, but rare methods reached the improvement of intrinsic NTE. In the present work, a significantly enhanced NTE is realized in Cu2P2O7 by applying low pressure. Especially, the volumetric coefficient of thermal expansion (CTE) of Cu2P2O7 reached to -50.0 × 10-6 K-1 (150-325K) under 0.25 GPa, which is increased by 47.5% compared to its NTE in a similar temperature range under atmosphere pressure. This character enables a more effective manifestation of the thermal compensation role of Cu2P2O7 in composites. The enhanced NTE mechanisms are analyzed by high pressure synchrotron X-ray diffraction, neutron diffraction at variable temperature and pressure, as well as density functional theory (DFT) calculations. The results show that applied pressure accelerates the contraction of the distance between adjacent CuO layers and CuO columns. Meanwhile, the low-frequency phonon contribution to NTE in α-Cu2P2O7 is improved. This work is meaningful for the exploration of methods to enhance NTE and the practical application of NTE materials.

Selective Dissolution of Calcium Pyrophosphate Dihydrate Crystals Using a Pyrophosphate Specific Receptor.

Pseudo-gout is caused by the deposition of highly insoluble calcium pyrophosphate dihydrate (CPPD) crystals in the joints of sufferers. This leads to inflammation and ultimately joint damage. The insolubility of CPPD is driven by the strong attraction of di-cationic calcium ions with tetra-anionic pyrophosphate ions. One of the challenges of dissolving CPPD is that a related mineral, hydroxy apatite (HA) is present in larger amounts in the form of bone and also contains strongly interacting calcium and phosphate ions. Our aim in this work was to selectively dissolve CPPD in preference to HA. To accomplish this, we used a known receptor for pyrophosphate that contains two complexed zinc ions that are ideally spaced to interact with the tetra-anion of pyrophosphate. We hypothesized that such a molecule could act as a preorganized tetra-cation that would be able to outcompete the two calcium ions present in the crystal lattice of CPPD. We demonstrate both visually and through analysis of released phosphorous that this molecule is able to preferentially dissolve CPPD over the closely related HA and thus can form the basis for a possible approach for the treatment of pseudo-gout.

Mono-ADP-ribosylation of peptides: an overview of synthetic and chemoenzymatic methodologies.

Adenosine diphosphate (ADP)-ribosylation is a ubiquitous post-translational modification that regulates vital biological processes like histone reorganization and DNA-damage repair through the modification of various amino acid residues. Due to advances in mass-spectrometry, the collection of long-known ADP-ribose (ADPr) acceptor sites, e.g. arginine, cysteine and glutamic acid, has been expanded with serine, tyrosine and histidine, among others. Well-defined ADPr-peptides are valuable tools for investigating the exact structures, mechanisms of action and interaction partners of the different flavors of this modification. This review provides a comprehensive overview of synthetic and chemoenzymatic methodologies that enabled the construction of peptides mono-ADP-ribosylated on various amino acids, and close mimetics thereof.

A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA.

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2'-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

The 6-phosphofructokinase reaction in Acetivibrio thermocellus is both ATP- and pyrophosphate-dependent.

Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

High frequency of BCP, but less CPP crystal-mediated calcification in cartilage and synovial membrane of osteoarthritis patients.

OBJECTIVE: Ectopic articular calcification is a common phenomenon of osteoarthritic joints, and closely related to disease progression. Identification of the involved calcium crystal types represents an important topic in research and clinical practice. Difficulties in accurate detection and crystal type identification have led to inconsistent data on the prevalence and spatial distribution of Basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) deposition. METHOD: Combining multiple imaging methods including conventional radiography, histology and Raman spectroscopy, this study provides a comprehensive analysis of BCP and CPP-based calcification, its frequency and distribution in cartilage and synovial membrane samples of 92 osteoarthritis patients undergoing knee replacement surgery. RESULTS: Conventional radiography showed calcifications in 35% of patients. Von Kossa staining detected calcified deposits in 88% and 57% of cartilage and synovial samples, respectively. BCP crystals presented as brittle deposits on top of the cartilage surface or embedded in synovial tissue. CPP deposits appeared as larger granular needle-shaped clusters or dense circular pockets below the cartilage surface or within synovial tissue. Spectroscopic analysis detected BCP crystals in 75% of cartilage and 43% of synovial samples. CPP deposition was only detected in 18% of cartilage and 15% of synovial samples, often coinciding with BCP deposits. CONCLUSION: BCP is the predominant crystal type in calcified cartilage and synovium while CPP deposition is rare, often coinciding with BCP. Distinct and qualitative information on BCP and CPP deposits in joint tissues gives rise to the speculation that different disease entities are involved that might need different treatment strategies.