ABSTRACT
The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described ß-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.IMPORTANCEAlgae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans. This bacterium uses the recently characterized ß-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions.
Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Glycolates , Paracoccus denitrificans , Paracoccus denitrificans/metabolism , Paracoccus denitrificans/genetics , Glycolates/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Metabolic Networks and Pathways/genetics , Glyoxylates/metabolism , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/genetics , Multigene FamilyABSTRACT
Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus, a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.
Subject(s)
Bacterial Proteins , Ethylene Glycol , Rhodococcus , Rhodococcus/metabolism , Rhodococcus/genetics , Ethylene Glycol/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Glycolates/metabolism , Glyoxylates/metabolism , Alcohol Dehydrogenase/metabolism , Alcohol Dehydrogenase/genetics , PeptidesABSTRACT
Climate warming poses a significant threat to global crop production and food security. However, our understanding of the molecular mechanisms governing thermoresponsive development in crops remains limited. Here we report that the auxiliary subunit of N-terminal acetyltransferase A (NatA) in rice OsNAA15 is a prerequisite for rice thermoresponsive growth. OsNAA15 produces two isoforms OsNAA15.1 and OsNAA15.2, via temperature-dependent alternative splicing. Among the two, OsNAA15.1 is more likely to form a stable and functional NatA complex with the potential catalytic subunit OsNAA10, leading to a thermoresponsive N-terminal acetylome. Intriguingly, while OsNAA15.1 promotes plant growth under elevated temperatures, OsNAA15.2 exhibits an inhibitory effect. We identified two glycolate oxidases (GLO1/5) as major substrates from the thermoresponsive acetylome. These enzymes are involved in hydrogen peroxide (H2O2) biosynthesis via glycolate oxidation. N-terminally acetylated GLO1/5 undergo their degradation through the ubiquitin-proteasome system. This leads to reduced reactive oxygen species (ROS) production, thereby promoting plant growth, particularly under high ambient temperatures. Conclusively, our findings highlight the pivotal role of N-terminal acetylation in orchestrating the glycolate-mediated ROS homeostasis to facilitate thermoresponsive growth in rice.
Subject(s)
Glycolates , Homeostasis , Oryza , Plant Proteins , Reactive Oxygen Species , Temperature , Oryza/metabolism , Oryza/growth & development , Oryza/drug effects , Oryza/genetics , Acetylation , Reactive Oxygen Species/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Glycolates/metabolism , Hydrogen Peroxide/metabolism , Proteolysis/drug effects , Gene Expression Regulation, Plant/drug effects , Alcohol Oxidoreductases/metabolismABSTRACT
Besides the historical and traditional use of nuclear magnetic resonance (NMR) spectroscopy as a structure elucidation tool for proteins and metabolites, its quantification ability allows the determination of metabolite amounts and therefore enzymatic activity measurements. For this purpose, 1H-NMR with adapted water pulse pre-saturation sequences and calibration curves with commercial standard solutions can be used to quantify the photorespiratory cycle intermediates, 2-phosphoglycolate and glycolate, associated with the phosphoglycolate phosphatase reaction. The intensity of the 1H-NMR signal of glycolate produced by the activity of purified recombinant Arabidopsis thaliana PGLP1 can therefore be used to determine PGLP1 enzymatic activities and kinetic parameters.
Subject(s)
Arabidopsis , Glycolates , Magnetic Resonance Spectroscopy , Phosphoric Monoester Hydrolases , Glycolates/metabolism , Glycolates/chemistry , Phosphoric Monoester Hydrolases/metabolism , Arabidopsis/metabolism , Arabidopsis/enzymology , Magnetic Resonance Spectroscopy/methods , Arabidopsis Proteins/metabolism , Enzyme Assays/methods , Kinetics , Recombinant Proteins/metabolismABSTRACT
Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.
Subject(s)
Alcohol Oxidoreductases , Arabidopsis , Phosphoric Monoester Hydrolases , Recombinant Proteins , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis/metabolism , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Phosphoric Monoester Hydrolases/genetics , Glycolates/metabolism , Enzyme Assays/methods , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Plant Leaves/metabolism , Plant Leaves/enzymology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Spectrophotometry/methodsABSTRACT
Amyotrophic lateral sclerosis (ALS) leads to death within 2-5 yr. Currently, available drugs only slightly prolong survival. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a supposedly central role of glycolic acid (GA) and D-lactic acid (DL)-both putative products of the Parkinson's disease associated glyoxylase DJ-1. Combined, not single, treatment with GA/DL restored axonal organelle phenotypes of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was not only accompanied by restoration of mitochondrial membrane potential but even dependent on it. Despite presenting an axonal transport deficiency as well, TDP43 patient-derived MNs did not share mitochondrial depolarization and did not respond to GA/DL treatment. GA and DL also restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites, recently reported being upstream of the mitochondrial phenotypes in FUS-ALS. Whereas these data point towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different neurodegenerative diseases characterized by mitochondrial depolarization.
Subject(s)
Amyotrophic Lateral Sclerosis , Glycolates , Lactic Acid , Mitochondria , Protein Deglycase DJ-1 , RNA-Binding Protein FUS , Superoxide Dismutase-1 , Humans , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/genetics , RNA-Binding Protein FUS/metabolism , RNA-Binding Protein FUS/genetics , Glycolates/metabolism , Glycolates/pharmacology , Mitochondria/metabolism , Protein Deglycase DJ-1/metabolism , Protein Deglycase DJ-1/genetics , Lactic Acid/metabolism , Superoxide Dismutase-1/metabolism , Superoxide Dismutase-1/genetics , Membrane Potential, Mitochondrial , Motor Neurons/metabolism , Lysosomes/metabolismABSTRACT
Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.
Subject(s)
Archaea , Ribulose-Bisphosphate Carboxylase , Ribulose-Bisphosphate Carboxylase/genetics , Ribulose-Bisphosphate Carboxylase/metabolism , Archaea/metabolism , Photosynthesis , Glycolates/metabolism , Phosphoric Monoester Hydrolases/metabolism , Oxygenases/metabolism , PentosesABSTRACT
Glycolic acid is widely employed in chemical cleaning, the production of polyglycolic acid-lactic acid, and polyglycolic acid. Currently, the bottleneck of glycolate biosynthesis lies on the imbalance of metabolic flux and the deficiency of NADPH. In this study, a dynamic regulation system was developed and optimized to enhance the metabolic flux from glucose to glycolate. Additionally, the knockout of transhydrogenase (sthA), along with the overexpression of pyridine nucleotide transhydrogenase (pntAB) and the implementation of the Entner-Doudoroff pathway, were performed to further increase the production of the NADPH, thereby increasing the titer of glycolate to 5.6 g/L. To produce glycolate from corn stover hydrolysate, carbon catabolite repression was alleviated and glucose utilization was accelerated. The final strain, E. coli Mgly10-245, is inducer-free, achieving a glycolate titer of 46.1 g/L using corn stover hydrolysate (77.1 % of theoretical yield). These findings will contribute to the advancement of industrial glycolate production.
Subject(s)
Escherichia coli , NADP Transhydrogenases , Escherichia coli/genetics , Escherichia coli/metabolism , Zea mays/metabolism , NADP/metabolism , Glycolates/metabolism , NADP Transhydrogenases/metabolism , Polyglycolic Acid/metabolism , Glucose/metabolism , Metabolic EngineeringABSTRACT
The physiology of different Escherichia coli stains was analyzed for growth with glycolate as a potentially promising sustainable sole source of carbon and energy. Different E. coli strains showed large differences regarding lag phases after provision of glycolate. Whereas E. coli W showed fast adaptation, E. coli BW25113, JM101, and BL21 (DE3) needed extensive time for adaption (up to 30 generations) until the attainable µmax was reached, which, at 30 °C, amounted to 0.20-0.25â¯h-1 for all strains. The overexpression of genes encoding glycolate degradation did neither overcome the need for adaptation of E. coli BL21 (DE3) nor improve growth of E. coli W. Rather, high level expression of proteins involved in uptake and initial degradation steps had an adverse effect on growth. Overall, the results show a promising capacity of E. coli strains for growth on glycolate.
Subject(s)
Carbon , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Carbon/metabolism , Glycolates/metabolismABSTRACT
Single-carbon (C1) biorefinery plays a key role in the consumption of global greenhouse gases and a circular carbon economy. Thereby, we have focused on the valorization of C1 compounds (e.g. methanol, formaldehyde, and formate) into multicarbon products, including bioplastic monomers, glycolate, and ethylene glycol. For instance, methanol, derived from the oxidation of CH4, can be converted into glycolate, ethylene glycol, or erythrulose via formaldehyde and glycolaldehyde, employing C1 and/or C2 carboligases as essential enzymes. Escherichia coli was engineered to convert formate, produced from CO via CO2 or from CO2 directly, into glycolate. Recent progress in the design of biotransformation pathways, enzyme discovery, and engineering, as well as whole-cell biocatalyst engineering for C1 biorefinery, was addressed in this review.
Subject(s)
Carbon , Methanol , Methanol/metabolism , Carbon/metabolism , Carbon Dioxide/metabolism , Ethylene Glycol/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Formates/metabolism , Formaldehyde/metabolism , Glycolates/metabolismABSTRACT
Fluroxypyr-meptyl (FLUME) is heterocyclic herbicide with internal absorption and transmission characteristics. Owing to its low cost and rapid efficacy, it has been widely used to control broad-leaved weeds in wheat, corn, and rice fields. However, the uptake, translocation, accumulation, and metabolism of FLUME in rice seedlings and the extent of oxidative stress induced by it remain largely unknown, which consequently restricts the comprehensive risk assessment of FLUME residues in the environment during rice production. Hence, we systematically investigated the growth and physiological responses of rice to FLUME and analyzed its uptake, translocation, accumulation, and metabolism in rice seedlings. The results indicated that under 0-0.12 mg/L FLUME treatment, only a small proportion of FLUME was translocated upward and accumulated in rice shoots following absorption via roots, with all the translocation factor values being < 1. Moreover, the distribution and enrichment ability of FLUME in rice seedlings were greater in roots than in shoots. Furthermore, we revealed that FLUME accumulation in rice seedlings evidently inhibited their growth and activated the defense system against oxidative stress, with an increase in the activity of antioxidant and detoxifying enzymes. In addition, multiple metabolic reactions of FLUME were observed in rice seedlings, including dehalogenation, hydroxylation, glycosylation, acetylation, and malonylation. Our study provides systematic insights into the uptake, translocation, accumulation, and metabolism of FLUME in rice seedlings as well as the oxidative stress induced by FLUME accumulation, which can help improve FLUME applications and environmental risk assessments in crops.
Subject(s)
Oryza , Seedlings , Seedlings/metabolism , Oryza/chemistry , Glycolates/analysis , Glycolates/metabolism , Oxidative Stress , Plant Roots/chemistryABSTRACT
Glycolate oxidase (GLO) is an FMN-containing enzyme localized in peroxisomes and performs in various molecular and biochemical mechanisms. It is a key player in plant glycolate and glyoxylate accumulation pathways. The role of GLO in disease and stress resistance is well-documented in various plant species. Although studies have been conducted regarding the role of GLO genes from spinach on a microbial level, the direct response of GLO genes to various stresses in short-season and leafy plants like lettuce has not been published yet. The genome of Lactuca sativa cultivar 'Salinas' (v8) was used to identify GLO gene members in lettuce by performing various computational analysis. Dual synteny, protein-protein interactions, and targeted miRNA analyses were conducted to understand the function of GLO genes. The identified GLO genes showed further clustering into two groups i.e., glycolate oxidase (GOX) and hydroxyacid oxidase (HAOX). Genes were observed to be distributed unevenly on three chromosomes, and syntenic analysis revealed that segmental duplication was prevalent. Thus, it might be the main reason for GLO gene diversity in lettuce. Almost all LsGLO genes showed syntenic blocks in respective plant genomes under study. Protein-protein interactions of LsGLO genes revealed various functional enrichments, mainly photorespiration, and lactate oxidation, and among biological processes oxidative photosynthetic carbon pathway was highly significant. Results of in-depth analyses disclosed the interaction of GLO genes with other members of the glycolate pathway and the activity of GLO genes in various organs and developmental stages in lettuce. The extensive genome evaluation of GLO gene family in garden lettuce is believed to be a reference for cloning and studying functional analyses of GLO genes and characterizing other members of glycolate/glyoxylate biosynthesis pathway in various plant species.
Subject(s)
Gardens , Lactuca , Lactuca/genetics , Lactuca/metabolism , Plants/metabolism , Glycolates/metabolism , GlyoxylatesABSTRACT
Diethylene glycol (DEG) mass poisonings have resulted from ingestion of adulterated pharmaceuticals, leading to proximal tubular necrosis and acute kidney injury. Diglycolic acid (DGA), one of the primary metabolites, accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to Krebs cycle intermediates such as succinate. Previous studies have shown that DGA is taken into kidney cells via the succinate-related dicarboxylate transporters. These studies have assessed whether the DGA that is taken up by primary cultures of human proximal tubule (HPT) cells is effluxed. In addition, a possible mechanism for efflux, via organic anion transporters (OATs) that exchange external organic anions for dicarboxylates inside the cell, was assessed using transformed cell lines that actively express OAT activities. When HPT cells were cultured on membrane inserts, then loaded with DGA and treated with the OAT4/5 substrate estrone sulfate or the OAT1/3 substrate para-aminohippurate, no DGA efflux was seen. A repeat of this experiment utilizing RPTEC/TERT1 cells with overexpressed OAT1 and OAT3 had similar results. In these cells, but not in HPT cells, co-incubation with succinate increased the uptake of PAH, confirming the presence of OAT activity in the RPTEC/TERT1 cells. Thus, despite OATs stimulation in cells with OAT activity, there was little to no efflux of DGA from the cells. This study concluded that DGA is poorly transported out of cells and that stimulation of OAT transporters is not a viable target for reducing DGA accumulation in cells.
Subject(s)
Glycolates , Kidney Tubules, Proximal , Rats , Humans , Animals , Kidney Tubules, Proximal/metabolism , Glycolates/toxicity , Glycolates/metabolism , Succinates/metabolism , Succinic Acid/metabolism , Organic Anion Transporters, Sodium-Independent/genetics , Organic Anion Transporters, Sodium-Independent/metabolismABSTRACT
As an alternative to chemical building blocks derived from algal biomass, the excretion of glycolate has been proposed. This process has been observed in green algae such as Chlamydomonas reinhardtii as a product of the photorespiratory pathway. Photorespiration generally occurs at low CO2 and high O2 concentrations, through the key enzyme RubisCO initiating the pathway via oxygenation of 1.5-ribulose-bisphosphate. In wild-type strains, photorespiration is usually suppressed in favour of carboxylation due to the cellular carbon concentrating mechanisms (CCMs) controlling the internal CO2 concentration. Additionally, newly produced glycolate is directly metabolized in the C2 cycle. Therefore, both the CCMs and the C2 cycle are the key elements which limit the glycolate production in wild-type cells. Using conventional crossing techniques, we have developed Chlamydomonas reinhardtii double mutants deficient in these two key pathways to direct carbon flux to glycolate excretion. Under aeration with ambient air, the double mutant D6 showed a significant and stable glycolate production when compared to the non-producing wild type. Interestingly, this mutant can act as a carbon sink by fixing atmospheric CO2 into glycolate without requiring any additional CO2 supply. Thus, the double-mutant strain D6 can be used as a photocatalyst to produce chemical building blocks and as a future platform for algal-based biotechnology. KEY POINTS: ⢠Chlamydomonas reinhardtii cia5 gyd double mutants were developed by sexual crossing ⢠The double mutation eliminates the need for an inhibitor in glycolate production ⢠The strain D6 produces significant amounts of glycolate with ambient air only.
Subject(s)
Chlamydomonas reinhardtii , Biotechnology , Carbon/metabolism , Carbon Dioxide/metabolism , Chlamydomonas reinhardtii/metabolism , Glycolates/metabolism , Photosynthesis , Plants/metabolismABSTRACT
Bisphosphoglycerate mutase (BPGM) is an erythrocyte-specific multifunctional enzyme that is responsible for the regulation of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells through its synthase and phosphatase activities; the latter enzymatic function is stimulated by the endogenous activator 2-phosphoglycolate (2-PG). 2,3-BPG is a natural allosteric effector of hemoglobin (Hb) that is responsible for decreasing the affinity of Hb for oxygen to facilitate tissue oxygenation. Here, crystal structures of BPGM with 2-PG in the presence and absence of 3-phosphoglycerate are reported at 2.25 and 2.48â Å resolution, respectively. Structure analysis revealed a new binding site for 2-PG at the dimer interface for the first time, in addition to the expected active-site binding. Also, conformational non-equivalence of the two active sites was observed as one of the sites was found in an open conformation, with the residues at the active-site entrance, including Arg100, Arg116 and Arg117, and the C-terminus disordered. The kinetic result is consistent with the binding of 2-PG to an allosteric or noncatalytic site as well as the active site. This study paves the way for the rational targeting of BPGM for therapeutic purposes, especially for the treatment of sickle cell disease.
Subject(s)
Bisphosphoglycerate Mutase , Glycolates , Binding Sites , Glycolates/metabolism , Phosphoric Monoester HydrolasesABSTRACT
Photorespiration accounts for 20-50 % reduction in grain yield in C3 crops. The process is essential to remove 2-phosphoglycolate produced due to the oxygenation activity of the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) enzyme. Attempts were made to improve photosynthesis through enriched CO2 concentration by installing numerous photorespiratory bypass modules in the chloroplast of several crops. In this study, we have introduced Escherichia coli glycolate catabolic pathway (ECGC) into rice chloroplast to bypass photorespiration partially (PB) or completely (FB). Five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and three subunits of glycolate dehydrogenase (GDH) were introduced to get FB plants, whereas only the three subunits of GDH were introduced to get PB plants. Southern analysis confirmed stable integration of the transgenes and their expression was confirmed by RT-qPCR analysis in the T3 progenies. Both FB and PB transformed lines exhibited increased photosynthetic efficiency, biomass, and grain yield than wild type (WT) with empty vector control. The introduction of ECGC pathway favoured the carboxylase activity of RuBisCO while decreasing its oxygenase activity fostering the functioning of Calvin-Benson cycle and resulting in an increased carbon-assimilation that was manifested in their superior architecture and harvest index. These findings will support rice and related cereal crop breeding programs to increase yield under elevated temperature and arid conditions.
Subject(s)
Chloroplasts/metabolism , Glycolates/metabolism , Metabolic Networks and Pathways/genetics , Oryza/growth & development , Oryza/genetics , Photosynthesis/physiology , Biomass , Crop Production , Crops, Agricultural/genetics , Crops, Agricultural/physiologyABSTRACT
Stroke is the second leading cause of death and disability worldwide. Current treatments, such as pharmacological thrombolysis or mechanical thrombectomy, reopen occluded arteries but do not protect against ischemia-induced damage that occurs before reperfusion or neuronal damage induced by ischemia/reperfusion. It has been shown that disrupting the conversion of glyoxal to glycolic acid (GA) results in a decreased tolerance to anhydrobiosis in Caenorhabditis elegans dauer larva and that GA itself can rescue this phenotype. During the process of desiccation/rehydration, a metabolic stop/start similar to the one observed during ischemia/reperfusion occurs. In this study, the protective effect of GA is tested in different ischemia models, i.e., in commonly used stroke models in mice and swine. The results show that GA, given during reperfusion, strongly protects against ischemic damage and improves functional outcome. Evidence that GA exerts its effect by counteracting the glutamate-dependent increase in intracellular calcium during excitotoxicity is provided. These results suggest that GA treatment has the potential to reduce mortality and disability in stroke patients.
Subject(s)
Brain Ischemia/drug therapy , Calcium/metabolism , Glycolates/pharmacology , Neuroprotective Agents/pharmacology , Reperfusion Injury/prevention & control , Animals , Brain Ischemia/metabolism , Desiccation , Disease Models, Animal , Glycolates/administration & dosage , Glycolates/metabolism , Male , Mice , Mice, Inbred C57BL , Neuroprotection/drug effects , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/metabolism , Reperfusion Injury/metabolism , SwineABSTRACT
Glycolate is a bulk chemical with wide applications in the textile, food processing, and pharmaceutical industries. Glycolate can be produced from glucose via the glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. However, two problems limit the productivity and yield of glycolate when using glucose as the sole carbon source. The first is a cofactor imbalance in the production of glycolate from glucose via the glycolysis pathway, since NADPH is required for glycolate production, while glycolysis generates NADH. To rectify this imbalance, the NADP+ -dependent glyceraldehyde 3-phosphate dehydrogenase GapC from Clostridium acetobutylicum was introduced to generate NADPH instead of NADH in the oxidation of glyceraldehyde 3-phosphate during glycolysis. The soluble transhydrogenase SthA was further eliminated to conserve NADPH by blocking its conversion into NADH. The second problem is an unfavorable carbon flux distribution between the tricarboxylic acid cycle and the glyoxylate shunt. To solve this problem, isocitrate dehydrogenase (ICDH) was eliminated to increase the carbon flux of glyoxylate and thereby improve the glycolate titer. After engineering through the integration of gapC, combined with the inactivation of ICDH, SthA, and by-product pathways, as well as the upregulation of the two key enzymes isocitrate lyase (encoding by aceA), and glyoxylate reductase (encoding by ycdW), the glycolate titer increased to 5.3 g/L with a yield of 1.89 mol/mol glucose. Moreover, an optimized fed-batch fermentation reached a titer of 41 g/L with a yield of 1.87 mol/mol glucose after 60 h.
Subject(s)
Escherichia coli , Glycolates , Metabolic Engineering/methods , Bacterial Proteins/genetics , Clostridium acetobutylicum/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Glucose/metabolism , Glycolates/analysis , Glycolates/metabolism , Metabolic Networks and Pathways/geneticsABSTRACT
Despite recent advancements in cisplatin (cis-diamminedichloroplatinum II) and other platinum-based chemotherapeutic drugs for treating solid tumors, their uses are limited by either in terms of toxicity and/or acquired drug resistance. These side effects have a dangerous problem with higher dose for severe patients. To overcome the low therapeutic ratio of the free drug, a polymeric nanoparticle drug delivery system has been explored promoting delivery of cisplatin to tumors. Recently, the applications of nanoparticles (NPs) have been underlined for encouraging the effects of chemotherapeutic drugs in cancerous cells. The intention of this project is to assess the potential of poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) for enhancing the effects of anticancer drug cisplatin. For the purpose, we have synthesized PLGA-cisplatin nanoparticles for increasing its bioavailability and studied the comparative cytotoxicity of free cisplatin and PLGA-cisplatin against MCF-7 cancer cell lines and HEK-293 normal cell lines. We have also analyzed the hallmarks of PLGA-cisplatin-induced apoptosis. The outcomes of this study may provide the possibility of delivery of anticancer drug to their specific site, which could minimize toxicity and optimize the drug efficacy.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Cisplatin/therapeutic use , Glycolates/metabolism , Polyesters/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cisplatin/pharmacology , Female , HumansABSTRACT
Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies, which is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multigene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep-well plate-scale high-throughput screening methods for the rapid screening of superior glycolate producers from a large library. A total of 22 gradient-strength promoter-5'-untranslated region (UTR) complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6 × 105 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/liter glycolate in a 5-liter bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multigene pathway optimizations. IMPORTANCE The use of strong promoters, such as pTrc and T7, to control gene expression not only needs the addition of expensive inducers but also results in excessive protein expression that may result in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of high-throughput screening methods restricted glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, and agar plate- and 48-well plate-scale high-throughput screening methods were then developed for the rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate-producing strain with good biosynthetic performance, and the use of the expensive inducer isopropyl-ß-d-thiogalactopyranoside (IPTG) was avoided, which broadens our understanding of the mechanism of glycolate synthesis.