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Journal of Basic Microbiology May 2024This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression...
This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression of biosynthetic enzyme genes, including phosphoglucomutase, hexokinase, phosphomannomutase, polysaccharide synthase, and UDP-glucose 4-epimerase genes. The study found higher EPS concentrations in seven recombinant strains compared to the wild-type C. militaris, indicating that the overexpression of key enzyme genes increased EPS production. Among them, the CM-pgm-2 strain exhibited the highest EPS production, reaching a concentration of 3.82 ± 0.26 g/L, which was 1.52 times higher than the amount produced by the wild C. militaris strain. Additionally, the regulatory effects of aromatic amino acids on the QS system of the CM-pgm-2 strain were investigated. Under the influence of 45 mg/L tryptophan, the EPS production in CM-pgm-2 reached 4.75 ± 0.20 g/L, representing a 1.90-fold increase compared to wild C. militaris strains. This study provided an effective method for the large-scale production of EPSs in C. militaris, and opened up new avenues for research into fungal QS mechanisms.
PubMed: 38771080
DOI: 10.1002/jobm.202400103 -
Biochemistry Jun 2024-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening....
-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.
Topics: Humans; Phosphoglucomutase; Saccharomyces cerevisiae; Proteolysis; Mutation, Missense; Ubiquitination; Congenital Disorders of Glycosylation; Protein Stability; Proteasome Endopeptidase Complex
PubMed: 38743592
DOI: 10.1021/acs.biochem.4c00042 -
ACS Omega May 2024Drought is the worst environmental stress constraint that inflicts heavy losses to global food production, such as wheat. The metabolic responses of seeds produced...
Drought is the worst environmental stress constraint that inflicts heavy losses to global food production, such as wheat. The metabolic responses of seeds produced overtransgenerational exposure to [CO] to recover drought's effects on wheat are still unexplored. Seeds were produced constantly for four generations (F1 to F4) under ambient CO ([CO], 400 μmol L) and elevated CO ([CO], 800 μmol L) concentrations, and then further regrown under natural CO conditions to investigate their effects on the stress memory metabolic processes liable for increasing drought resistance in the next generation (F5). At the anthesis stage, plants were subjected to normal (100% FC, field capacity) and drought stress (60% FC) conditions. Under drought stress, plants of transgenerational [CO] exposed seeds showed markedly increased superoxide dismutase (16%), catalase (24%), peroxidase (9%), total antioxidants (14%), and proline (35%) levels that helped the plants to sustain normal growth through scavenging of hydrogen peroxide (11%) and malondialdehyde (26%). The carbohydrate metabolic enzymes such as aldolase (36%), phosphoglucomutase (12%), UDP-glucose pyrophosphorylase (25%), vacuolar invertase (33%), glucose-6-phosphate-dehydrogenase (68%), and cell wall invertase (17%) were decreased significantly; however, transgenerational seeds produced under [CO] showed a considerable increase in their activities in drought-stressed wheat plants. Moreover, transgenerational [CO] exposed seeds under drought stress caused a marked increase in leaf Ψ (15%), chlorophyll (19%), chlorophyll (8%), carotenoids (12%), grain spike (16%), hundred grain weight (19%), and grain yield (10%). Hence, transgenerational seeds exposed to [CO] upregulate the drought recovery metabolic processes to improve the grain yield of wheat under drought stress conditions.
PubMed: 38737051
DOI: 10.1021/acsomega.3c10379 -
The Israel Medical Association Journal... May 2024
Topics: Humans; Female; Pregnancy; Infant, Newborn; Adult; Pregnancy Outcome; Glycogen Storage Disease; Pregnancy Complications
PubMed: 38736348
DOI: No ID Found -
The New Phytologist Jul 2024Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO and glucose. The switch between these trophic modes and the role of...
Glycogen synthesis prevents metabolic imbalance and disruption of photosynthetic electron transport from photosystem II during transition to photomixotrophy in Synechocystis sp. PCC 6803.
Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O evolution and CO uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.
Topics: Synechocystis; Glycogen; Photosynthesis; Electron Transport; Photosystem II Protein Complex; Mutation; Glucose; Carbon Dioxide; Oxygen; Glucose-1-Phosphate Adenylyltransferase; Phosphoglucomutase
PubMed: 38706429
DOI: 10.1111/nph.19793 -
Journal of Microbiology and... May 2024Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for...
Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from and the endogenous overexpression of genes encoding UDP-glucose pyrophosphorylase and encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.
Topics: Corynebacterium glutamicum; Apigenin; Metabolic Engineering; Glucosides; Glycosylation; Bacillus licheniformis; Uridine Diphosphate Glucose; Bacterial Proteins; UTP-Glucose-1-Phosphate Uridylyltransferase; Glycosyltransferases
PubMed: 38563097
DOI: 10.4014/jmb.2401.01017 -
Journal of Plant Physiology May 2024Roots exhibit hydrotropism in response to moisture gradients, with the hydrotropism-related gene Mizu-kussei1 (MIZ1) playing a role in regulating root hydrotropism in an...
Roots exhibit hydrotropism in response to moisture gradients, with the hydrotropism-related gene Mizu-kussei1 (MIZ1) playing a role in regulating root hydrotropism in an oblique orientation. However, the mechanisms underlying MIZ1-regulated root hydrotropism are not well understood. In this study, we employed obliquely oriented experimental systems to investigate root hydrotropism in Arabidopsis. We found that the miz1 mutant displays reduced root hydrotropism but increased root gravitropism following hydrostimulation, as compared to wild-type plants. Conversely, overexpression of AtMIZ1 leads to enhanced root hydrotropism but decreased root gravitropism following hydrostimulation, as compared to wild-type plants. Using co-immunoprecipitation followed by mass spectrometry (IP-MS), we explored proteins that interact with AtMIZ1, and we identified PGMC1 co-immunoprecipitated with MIZ1 in vivo. Furthermore, the miz1 mutant exhibited higher expression of the PGMC1 gene and increased phosphoglucomutase (PGM) activity, while AtMIZ1 overexpressors resulted in lower expression of the PGMC1 gene, reduced amyloplast amount, and reduced PGM activity in comparison to wild-type roots. In addition, different Arabidopsis natural accessions having difference in their hydrotropic response demonstrated expression level of PGMC1 was negatively correlated with hydrotropic root curvature and AtMIZ1 expression. Our results provide valuable insights into the role of amyloplast in MIZ1-regulated root hydrotropism.
Topics: Arabidopsis; Arabidopsis Proteins; Water; Tropism; Gravitropism; Plant Roots
PubMed: 38507925
DOI: 10.1016/j.jplph.2024.154224 -
Protein Science : a Publication of the... Apr 2024Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and...
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β-phosphoglucomutase (β-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β-PGM activity and hence, Rv3400 encodes for β-PGM in Mtb. Our data also confirm that Mtb β-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β-PGM converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β-PGM in the physiology of Mtb.
Topics: Phosphoglucomutase; Maltose; Mycobacterium tuberculosis; Trehalose; Phosphates; Glucose
PubMed: 38501428
DOI: 10.1002/pro.4943 -
BMC Infectious Diseases Feb 2024Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is...
BACKGROUND
Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms.
METHOD
After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured.
RESULTS
GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum.
CONCLUSION
The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.
Topics: Humans; Leishmania infantum; Isoenzymes; Antioxidants; Glutathione Peroxidase; Superoxide Dismutase
PubMed: 38360592
DOI: 10.1186/s12879-024-09069-7 -
PloS One 2024The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal...
The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.
Topics: Cordyceps; RNA, Ribosomal, 18S; Gene Expression Profiling; Genes, Plant; Real-Time Polymerase Chain Reaction; Reference Standards; Tubulin; Ubiquitin
PubMed: 38319940
DOI: 10.1371/journal.pone.0287882