Metabolic Reprogramming in Sickle Cell Diseases: Pathophysiology and Drug Discovery Opportunities.

Sickle cell disease (SCD) is a genetic disorder that affects millions of individuals worldwide. Chronic anemia, hemolysis, and vasculopathy are associated with SCD, and their role has been well characterized. These symptoms stem from hemoglobin (Hb) polymerization, which is the primary event in the molecular pathogenesis of SCD and contributes to erythrocyte or red blood cell (RBC) sickling, stiffness, and vaso-occlusion. The disease is caused by a mutation at the sixth position of the ß-globin gene, coding for sickle Hb (HbS) instead of normal adult Hb (HbA), which under hypoxic conditions polymerizes into rigid fibers to distort the shapes of the RBCs. Only a few therapies are available, with the universal effectiveness of recently approved therapies still being monitored. In this review, we first focus on how sickle RBCs have altered metabolism and then highlight how this understanding reveals potential targets involved in the pathogenesis of the disease, which can be leveraged to create novel therapeutics for SCD.

The cell structure damage and embden-meyerhof-parnas pathway inhibition of Listeria monocytogenes induced by glycinin basic peptide.

Glycinin basic peptide (GBP) is a natural antibacterial peptide. This study aimed to explore the antibacterial characteristics of GBP against Listeria monocytogenes (L. monocytogenes) by measuring the membrane potential, membrane permeability, cell damage, morphological changes, respiration metabolism inhibition and DNA content. GBP increased the surface zeta potential and decreased the trans-membrane potential of L. monocytogenes in a dose-dependent manner. Compared with the control, the electrical conductivities of GBP-treated bacterial suspensions were significantly increased. The percentages of bacteria with damaged membrane increased from 6.40% to 70.90% with GBP from 0 to 0.8 mg/mL. Obvious rupture and deform of bacterial cells with GBP were observed by transmission electron microscope (TEM), showing the destructive effect of GBP on L. monocytogenes. GBP also inhibited the embden-meyerhof-parnas pathway of the bacterial respiration metabolism and reduced the activities of its key regulator enzymes. Besides, the content of DNA in GBP-treated L. monocytogenes was lower than that in control.

Alternative carbohydrate pathways - enzymes, functions and engineering.

Metabolic engineering is crucial in the development of production strains for platform chemicals, pharmaceuticals and biomaterials from renewable resources. The central carbon metabolism (CCM) of heterotrophs plays an essential role in the conversion of biomass to the cellular building blocks required for growth. Yet, engineering the CCM ultimately aims toward a maximization of flux toward products of interest. The most abundant dissimilative carbohydrate pathways amongst prokaryotes (and eukaryotes) are the Embden-Meyerhof-Parnas (EMP) and the Entner-Doudoroff (ED) pathways, which build the basics for heterotrophic metabolic chassis strains. Although the EMP is regarded as the textbook example of a carbohydrate pathway owing to its central role in production strains like Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis, it is either modified, complemented or even replaced by alternative carbohydrate pathways in different organisms. The ED pathway also plays key roles in biotechnological relevant bacteria, like Zymomonas mobilis and Pseudomonas putida, and its importance was recently discovered in photoautotrophs and marine microorganisms. In contrast to the EMP, the ED pathway and its variations are not evolutionary optimized for high ATP production and it differs in key principles such as protein cost, energetics and thermodynamics, which can be exploited in the construction of unique metabolic designs. Single ED pathway enzymes and complete ED pathway modules have been used to rewire carbon metabolisms in production strains and for the construction of cell-free enzymatic pathways. This review focuses on the differences of the ED and EMP pathways including their variations and discusses the use of alternative pathway strategies for in vivo and cell-free metabolic engineering.

The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.

Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner-Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO2 via the Calvin-Benson cycle.

Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Xanthomonas campestris pv. campestris.

Xanthomonads are plant pathogenic proteobacteria that produce the polysaccharide xanthan. They are assumed to catabolize glucose mainly via the Entner-Doudoroff pathway. Whereas previous studies have demonstrated no phosphofructokinase (PFK) activity in xanthomonads, detailed genome analysis revealed in Xanthomonas campestris pathovar campestris (Xcc) genes for all Embden-Meyerhof-Parnas pathway (glycolysis) enzymes, including a conserved pfkA gene similar to 6-phosphofructokinase genes. To address this discrepancy between genetic and physiological properties, the pfkA gene of Xcc strain B100 was cloned into the expression vector pET28a+. The 45-kDa pfkA gene product exhibited no conventional PFK activity. Bioinformatic analysis of the Xcc PfkA amino acid sequence suggested utilization of pyrophosphate as an alternative cosubstrate. Pyrophosphate-dependent PFK activity was shown in an in vitro enzyme assay for purified Xcc PfkA, as well as in the Xcc B100 crude protein extract. Kinetic constants were determined for the forward and reverse reactions. Primary structure conservation indicates the global presence of similar enzymes among Xanthomonadaceae.

Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron-transfer functionality of the natural cofactors, NAD(P)+, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN+-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN+. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN+-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ~3.4 × 106-fold switch in cofactor specificity from its native cofactor NADP+ to NMN+. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN+, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN+-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha.

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P PDH , P PYK , P FBA , P PGM , P GLK , P TRI , P GPI , P ADH1 , P TEF1 and P GCW14 ) were obtained with the activity range of 13%-130% of the common promoter P GAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P PDH and P GCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P ICL1 , rhamnose-induced P LRA3 and P LRA4 , and a bidirectional promoter (P Mal -P Per ) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P LRA3 by 4.7-10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

The Inactivation of Enzymes Belonging to the Central Carbon Metabolism Is a Novel Mechanism of Developing Antibiotic Resistance.

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes.IMPORTANCE Antibiotic resistance has been largely considered a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated on mechanisms that affect the antibiotics (mutations in transporters, efflux pumps, and antibiotic-modifying enzymes, or their regulators) or their targets (i.e., target mutations, protection, or bypass). Usually, antibiotic resistance-associated metabolic changes were considered a consequence (fitness costs) and not a cause of antibiotic resistance. Herein, we show that alterations in the central carbon bacterial metabolism can also be the cause of antibiotic resistance. In the study presented here, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas pathway. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that antibiotic resistance can be under metabolic control, our results provide evidence that antibiotic resistance is intertwined with the bacterial metabolism.

Effects of heat shock treatment on the survival rate of Lactobacillus acidophilus after freeze-drying.

Herein we investigated the effects of heat shock treatment on the resistance of Lactobacillus acidophilus ATCC4356 to freeze-drying and the underlying mechanisms. We assessed the survival rate, cell morphology, enzyme activities, and metabolites in glycometabolism and energy metabolism. Heat shock treated at 45 °C for 30 min has increased the survival rate from 39.1% to 56.3% and had a certain protective effect on the integrity of the cell wall and membrane after freeze-drying. Activities of key enzymes, namely glucose-6-phosphate isomerase and lactate dehydrogenase in the glycolytic pathway; phosphoglucomutase, UDP-glucose pyrophosphorylase, and glycosyltransferases in the glycogen biosynthetic pathway; and Na+ -K+ -ATPase in energy metabolism were significantly altered. Further, the utilization rate of extracellular glucose in the broth decreased 7.59% but the conversion rate of intracellular glucose increased 24.04%, which led to the production of lactic acid and energy. Meanwhile, the production of polysaccharides with potential protectant function was increased by 47.6% and the proportion of glucose in the monosaccharide fraction decreased from 21% to 17%. However, the production of galactose increased from 17% to 26%, consequently enhancing the activities and survival rate of bacterial cells in a freeze-drying environment. This is the first study to determine the potential mechanisms and metabolic changes induced by heat shock treatment that make LAB tolerant to freeze-drying, and providing a new insight on the anti-adversity for LAB during the process.

Glycolytic Shunts Replenish the Calvin-Benson-Bassham Cycle as Anaplerotic Reactions in Cyanobacteria.

The recent discovery of the Entner-Doudoroff (ED) pathway as a third glycolytic route beside Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (OPP) pathway in oxygenic photoautotrophs requires a revision of their central carbohydrate metabolism. In this study, unexpectedly, we observed that deletion of the ED pathway alone, and even more pronounced in combination with other glycolytic routes, diminished photoautotrophic growth in continuous light in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, we found that the ED pathway is required for optimal glycogen catabolism in parallel to an operating Calvin-Benson-Bassham (CBB) cycle. It is counter-intuitive that glycolytic routes, which are a reverse to the CBB cycle and do not provide any additional biosynthetic intermediates, are important under photoautotrophic conditions. However, observations on the ability to reactivate an arrested CBB cycle revealed that they form glycolytic shunts that tap the cellular carbohydrate reservoir to replenish the cycle. Taken together, our results suggest that the classical view of the CBB cycle as an autocatalytic, completely autonomous cycle that exclusively relies on its own enzymes and CO2 fixation to regenerate ribulose-1,5-bisphosphate for Rubisco is an oversimplification. We propose that in common with other known autocatalytic cycles, the CBB cycle likewise relies on anaplerotic reactions to compensate for the depletion of intermediates, particularly in transition states and under fluctuating light conditions that are common in nature.

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4.

Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

Glycolysis as the Central Core of Fermentation.

The increasing concerns of greenhouse gas emissions have increased the interest in dark fermentation as a means of productions for industrial chemicals, especially from renewable cellulosic biomass. However, the metabolism, including glycolysis, of many candidate organisms for cellulosic biomass conversion through consolidated bioprocessing is still poorly understood and the genomes have only recently been sequenced. Because a variety of industrial chemicals are produced directly from sugar metabolism, the careful understanding of glycolysis from a genomic and biochemical point of view is essential in the development of strategies for increasing product yields and therefore increasing industrial potential. The current review discusses the different pathways available for glycolysis along with unexpected variations from traditional models, especially in the utilization of alternate energy intermediates (GTP, pyrophosphate). This reinforces the need for a careful description of interactions between energy metabolites and glycolysis enzymes for understanding carbon and electron flux regulation.