-
ChemSusChem Mar 2023A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag...
A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag and dockerin-cohesin) and enzyme component self-oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher-SpyTag and cohesins are prepared to recruit the four dockerin-containing cascade enzymes (i. e., alpha-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol 1-phosphatase) that can convert starch into inositol, forming multi-enzyme complexes. These self-assembled enzyme complexes show higher initial reaction rates than the four-enzyme cocktail. Moreover, water-insoluble self-assembled multi-enzyme complexes are observed, being the carrier-free immobilized multi-enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier-free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.
Topics: Enzymes, Immobilized; Multienzyme Complexes; Peptides; Inositol
PubMed: 36538347
DOI: 10.1002/cssc.202202153 -
BMC Plant Biology Dec 2022Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly...
BACKGROUND
Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly understood. To elucidate the biosynthesis mechanism of ginseng polysaccharides, combined the transcriptome analysis and polysaccharides content determination were performed on the roots, stems, and leaves collected from four cultivars of ginseng.
RESULTS
The results indicated that the total contents of nine monosaccharides were highest in the roots. Moreover, the total content of nine monosaccharides in the roots of the four cultivars were different but similar in stems and leaves. Glucose (Glc) was the most component of all monosaccharides. In total, 19 potential enzymes synthesizing of ginseng polysaccharides were identified, and 17 enzymes were significantly associated with polysaccharides content. Among these genes, the expression of phosphoglucomutase (PGM), glucose-6-phosphate isomerase (GPI), UTP-glucose-1-phosphate uridylyltransferase (UGP2), fructokinase (scrK), mannose-1-phosphate guanylyltransferase (GMPP), phosphomannomutase (PMM), UDP-glucose 4-epimerase (GALE), beta-fructofuranosidase (sacA), and sucrose synthase (SUS) were correlated with that of MYB, AP2/ERF, bZIP, and NAC transcription factors (TFs). These TFs may regulate the expression of genes involved in ginseng polysaccharides synthesis.
CONCLUSION
Our findings could provide insight into a better understanding of the regulatory mechanism of polysaccharides biosynthesis, and would drive progress in genetic improvement and plantation development of ginseng.
Topics: Transcriptome; Panax; Gene Expression Profiling; Polysaccharides; Monosaccharides
PubMed: 36529733
DOI: 10.1186/s12870-022-03995-x -
Journal of Experimental Botany Mar 2023Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and...
Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and glucose-6-phosphate by phosphoglucomutases connects the metabolism of these polysaccharides with central carbon metabolism. However, knowledge about how this connection affects the ability of cells to cope with environmental stresses is still scarce. The cyanobacterium Synechocystis sp. PCC 6803 has two enzymes with phosphoglucomutase activity, PGM (phosphoglucomutase) and PMM/PGM (phosphomannomutase/phosphoglucomutase). In this work, we generated a null mutant of PGM (∆PGM) that exhibits very reduced phosphoglucomutase activity (1% of wild type activity). Although this mutant accumulates moderate amounts of glycogen, its phenotype resembles that of glycogen-less mutants, including high light sensitivity and altered response to nitrogen deprivation. Using an on/off arsenite promoter, we demonstrate that PMM/PGM is essential for growth and responsible for the remaining phosphoglucomutase activity in the ∆PGM strain. Furthermore, overexpression of PMM/PGM in the ∆PGM strain is enough to revoke the phenotype of this mutant. These results emphasize the importance of an adequate flux between glycogen and central carbon metabolism to maintain cellular fitness and indicate that although PGM is the main phosphoglucomutase activity, the phosphoglucomutase activity of PMM/PGM can substitute it when expressed in sufficient amounts.
Topics: Phosphoglucomutase; Glycogen; Carbon; Starch; Cyanobacteria
PubMed: 36454663
DOI: 10.1093/jxb/erac474 -
Journal of Inherited Metabolic Disease Jan 2023
Topics: Humans; Glycosylation; Phosphotransferases (Phosphomutases); Mutation; Congenital Disorders of Glycosylation; Phosphoglucomutase
PubMed: 36451346
DOI: 10.1002/jimd.12576 -
Computational and Structural... 2022The unique biological and rheological properties make hyaluronic acid a sought-after material for medicine and cosmetology. Due to very high purity requirements for... (Review)
Review
The unique biological and rheological properties make hyaluronic acid a sought-after material for medicine and cosmetology. Due to very high purity requirements for hyaluronic acid in medical applications, the profitability of streptococcal fermentation is reduced. Production of hyaluronic acid by recombinant systems is considered a promising alternative. Variations in combinations of expressed genes and fermentation conditions alter the yield and molecular weight of produced hyaluronic acid. This review is devoted to the current state of hyaluronic acid production by recombinant bacterial and fungal organisms.
PubMed: 36420162
DOI: 10.1016/j.csbj.2022.11.013 -
Carbohydrate Polymers Jan 2023A major challenge to large-scale production and utilization of bacterial cellulose (BC) for various applications is its low yield and productivity by bacterial cells and...
A major challenge to large-scale production and utilization of bacterial cellulose (BC) for various applications is its low yield and productivity by bacterial cells and the high cost of feedstock. A supplementation of the classical expensive Hestrin and Schramm (HS) medium with 1 % polyethylene terephthalate ammonia hydrolysate (PETAH) resulted in 215 % high yield. Although the physicochemical properties of BC were not significantly influenced, the BC produced in 1 % PETAH-supplemented HS medium showed a higher surface area, which showed 1.39 times higher adsorption capacity for tetracycline than BC produced in HS medium. The 1 % PETAH-supplemented HS medium respectively enhanced the activity of α-UDP-glucose pyrophosphorylase and α-phosphoglucomutase by 30.63 % and 135.24 % and decreased the activity of pyruvate kinase and phosphofructokinase by 40.34 % and 52.63 %. The results of this study provide insights into the activation mechanism of Taonella mepensis by PETAH supplementation for high yield and productivity of BC.
Topics: Gluconacetobacter xylinus; Cellulose; Polyethylene Terephthalates; Culture Media
PubMed: 36372499
DOI: 10.1016/j.carbpol.2022.120301 -
Pathogens (Basel, Switzerland) Oct 2022In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen...
In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 μM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 μM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on 's galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.
PubMed: 36297231
DOI: 10.3390/pathogens11101174 -
Bioengineering (Basel, Switzerland) Oct 2022The polysaccharides found in Lentinula edodes have a variety of medicinal properties, such as anti-tumor and anti-viral effects, but their content in L. edodes...
The polysaccharides found in Lentinula edodes have a variety of medicinal properties, such as anti-tumor and anti-viral effects, but their content in L. edodes sporophores is very low. In this study, Fe2+ was added to the liquid fermentation medium of L. edodes to analyze its effects on mycelial growth, polysaccharide and enzyme production, gene expression, and the activities of enzymes involved in polysaccharide biosynthesis, and in vitro antioxidation of polysaccharides. The results showed that when 200 mg/L of Fe2+ was added, with 7 days of shaking at 150 rpm and 3 days of static culture, the biomass reached its highest value (0.28 mg/50 mL) 50 days after the addition of Fe2+. Besides, Fe2+ addition also enhanced intracellular polysaccharide (IPS) and exopolysaccharide (EPS) productions, the levels of which were 2.98- and 1.79-fold higher than the control. The activities of the enzymes involved in polysaccharides biosynthesis, including phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), and UDPG-pyrophosphorylase (UGP) were also increased under Fe2+ addition. Maximum PGI activity reached 1525.20 U/mg 30 days after Fe2+ addition, whereas PGM and UGP activities reached 3607.05 U/mg and 3823.27 U/mg 60 days after Fe2+ addition, respectively. The Pearson correlation coefficient showed a strong correlation (p < 0.01) between IPS production and PGM and UGP activities. The corresponding coding genes of the three enzymes were also upregulated. When evaluating the in vitro antioxidant activities of polysaccharides, EPS from all Fe2+-treated cultures exhibited significantly better capacity (p < 0.05) for scavenging -OH radicals. The results of the two-way ANOVA indicated that the abilities of polysaccharides to scavenge O2− radicals were significantly (p < 0.01) affected by Fe2+ concentration and incubation time. These results indicated that the addition of iron provided a good way to achieve desirable biomass, polysaccharide production, and the in vitro antioxidation of polysaccharides from L. edodes.
PubMed: 36290549
DOI: 10.3390/bioengineering9100581 -
Journal of Thoracic Disease Sep 2022Lung adenocarcinoma (LUAD) is the most important subtype of lung cancer and usually metastasizes. Patients with LUAD usually had a poor prognosis. Identifying viable...
High expression of glycolysis-related gene in relation to poor prognosis and deficient immune cells infiltration in lung adenocarcinoma: a study based on bioinformatics analysis.
BACKGROUND
Lung adenocarcinoma (LUAD) is the most important subtype of lung cancer and usually metastasizes. Patients with LUAD usually had a poor prognosis. Identifying viable molecular markers for diagnostic and prognostic prediction among individuals with LUAD is critical for the future management of this disease. This study aimed to determine and verify a correlation between the glycolysis-related phosphoglucomutase 2 () gene and dissatisfactory results and deficient infiltration of immune cells in LUAD.
METHODS
The expression of in LUAD and adjoining normal tissues was screened from The Cancer Genome Atlas (TCGA) data and human protein atlas (HPA), and validatied by quantative reverse transcription polymerase chain reaction (qRT-PCR). We examined the correlation between expression and clinicopathologic characteristics (including pathological stage, gender, M stage, smoker, age, N stage, race, and number pack years smoked) by multivariable approaches and Kaplan-Meier survival curves. The proteins network with was built using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The correlation between expression and infiltration of immune cells, along with the corresponding gene marker sets, was investigated through the Gene Expression Profiling Interactive Analysis (GEPIA) and Tumor Immune Estimation Resource (TIMER) databases. We evaluated the possible correlation between expression and progression-free interval (PFI), disease-specific survival (DSS), and overall survival (OS) in LUAD patients.
RESULTS
Expression of was up-regulated in LUAD tissues (P=0.003). According to the multivariate logistic regression analysis, the elevated expression level of PGM2 exhibited a remarkable correlation with advanced tumor-node-metastasis (TNM) stage, high-grade malignancy, and primary therapeutic outcome . Overexpression of was shown to be correlated with an unfavorable prognosis including OS (P=0.004, HR =1.54), DSS (P=0.003, HR =1.77), and PFI (P=0.003, HR =1.5) in LUAD. The proteins PGM1 and UGP2 were shown to have a significant correlation with . Additionally, was associated with the lack of infiltrating immune cells as well as their associated gene marker sets in LUAD.
CONCLUSIONS
Overexpression of was shown to be associated with the progression and an unfavorable prognosis of LUAD, as well as with inefficient immune cell infiltration. was expected to be a potential biological marker for predicting the prognosis of patients with LUAD.
PubMed: 36245582
DOI: 10.21037/jtd-22-1043 -
ELife Oct 2022The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene which affect protein -linked glycosylation. The...
The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene which affect protein -linked glycosylation. The yeast gene encodes a homolog of human . We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, V238M and -F126L, or wild-type . We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including , which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the -V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the -V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles.
Topics: Humans; Saccharomyces cerevisiae; Congenital Disorders of Glycosylation; Phosphoglucomutase; Mutant Proteins; Saccharomyces cerevisiae Proteins
PubMed: 36214454
DOI: 10.7554/eLife.79346