ABSTRACT
Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by â¼45 %, while metformin reduced cGPDH and mGPDH activities by â¼32 % and â¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed â¼50 % and â¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.
Subject(s)
Adipocytes , Genistein , Glucose , Glycerol Kinase , Glycerolphosphate Dehydrogenase , Hepatocytes , Metformin , Genistein/pharmacology , Metformin/pharmacology , Mice , Animals , Glycerol Kinase/metabolism , Glycerol Kinase/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Adipocytes/drug effects , Adipocytes/metabolism , Glycerolphosphate Dehydrogenase/metabolism , Glycerolphosphate Dehydrogenase/genetics , Glucose/metabolism , 3T3-L1 Cells , Hep G2 Cells , Glycerophosphates/metabolism , Glycerophosphates/pharmacology , KineticsABSTRACT
ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.
Subject(s)
Adenosine Triphosphate , Arginine , Adenosine Triphosphate/metabolism , Arginine/metabolism , Artificial Cells/metabolism , Glycerophosphates/metabolism , Glycerol Kinase/metabolism , Glycerol Kinase/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Lipids/biosynthesis , Phospholipids/metabolism , Metabolic Networks and PathwaysABSTRACT
Triglyceride (TG) metabolism is a key factor that affects residual feed intake (RFI); however, few studies have been conducted on the related gene expression in poultry. The aim of the present study was to investigate the expression of genes and their associations with RFI in meat-type ducks. Weight gain and feed intake (FI) at an age 21-42 days were measured and the RFI was calculated. Quantitative PCR was used to test the expression of the six identified genes, namely peroxisome proliferator activated receptor γ (PPARγ), glycerol kinase 2 (GK2), glycerol-3-phosphate dehydrogenase 1 (GPD1), glycerol kinase (GYK), lipase E (LIPE), and lipoprotein lipase (LPL) in the duodenum in the high RFI (HRFI) and low RFI (LRFI) groups. The results demonstrated that daily feed intake, feed conversion ratio (FCR), and RFI were markedly higher in HRFI ducks than those in LRFI ducks. Moreover, the levels of expression of PPARγ, GK2, and LIPE were significantly higher in the LRFI group than those in the HRFI group. Correlation analysis showed that PPARγ, GK2, and LIPE were significantly negatively associated with FCR and RFI. Furthermore, gene expression levels were negatively associated with the measured phenotype. The association of GK2 with PPARγ, GPD1, LPL, and LIPE was positive. The relationship between the TG related gene and RFI was further verified to potentially develop pedigree poultry breeding programs. The results of this study suggested that the expression of genes correlated with TG metabolism and transport is up-regulated in the duodenum of ducks with high feed efficiency. PPARγ, GK2, and LIPE are important genes that affect RFI. The results of the present study provide information that could facilitate further explorations of the mechanism of RFI and potential markers at the molecular and cellular levels.
Subject(s)
Ducks , Glycerol Kinase , Animals , Ducks/genetics , Glycerol Kinase/genetics , PPAR gamma/genetics , Animal Feed/analysis , Eating/genetics , Meat/analysis , Gene Expression , TriglyceridesABSTRACT
The contiguous gene deletion syndromes (CGDS) are rare genomic disorders resulting from the deletion of large segments of DNA, manifested as the concurrence of apparently unrelated clinical features. A typical example of CGDS is Xp21 contiguous gene deletion syndrome that involves GK and its neigh-boring genes (usually DMD and NR0B1) and results in a complex phenotype, which is related to the size of deletion and involved genes. Development delay and intellectual disability are almost a constant feature of patients with CGDS. We report the case of a boy with Duchenne muscular dystrophy (DMD) and glycerol kinase deficiency (GKD) as part of the contiguous gene deletion syndrome Xp2.1, in association with intellectual disability (ID) in whom multiplex ligation-dependent probe amplification (MLPA) test first identified a hemizygous deletion involving the entire dystrophin gene. Subsequently, the array CGH study identified a maternally inherited hemizygous deletion of the Xp21.2-Xp21.1 region of approximately 3.7Mb that included both DMD and GK genes confirming the diagnosis of Xp21 CGDS. Moreover, we report a review of the cases published in the literature over the last 20 years, for which a better description of the genes involved in the syndrome was available. Intellectual disability does not appear as a constant feature of the syndrome, reiterating the concept that complex GKD syndrome results from small deletions that affect closely related but separate loci for DMD, GK and adrenal hypoplasia, rather than a single large deletion including all genes. This case highlights the importance of more in-depth genetic investigations in presence of apparently unrelated clinical findings, allowing an accurate diagnosis of contiguous gene deletion syndromes.
Subject(s)
Intellectual Disability , Muscular Dystrophy, Duchenne , Humans , Muscular Dystrophy, Duchenne/complications , Muscular Dystrophy, Duchenne/diagnosis , Muscular Dystrophy, Duchenne/genetics , Hypoadrenocorticism, Familial/genetics , Intellectual Disability/diagnosis , Intellectual Disability/genetics , Glycerol Kinase/genetics , Gene DeletionABSTRACT
BACKGROUND: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. METHODS: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. RESULTS: The overexpressed bioactive His-GK was purified apparently to homogeneity (â¼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of â¼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. CONCLUSION: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.
Subject(s)
Diabetes Mellitus, Type 2 , Glycerol Kinase , Humans , Glycerol Kinase/genetics , Glycerol Kinase/chemistry , Glycerol Kinase/metabolism , Glycerol , Molecular Chaperones/genetics , Escherichia coliABSTRACT
Pseudomonas aeruginosa is a Gram-negative bacterium capable of widespread niches, which is also one of the main bacteria that cause patient infection. The metabolic diversity of Pseudomonas aeruginosa is an essential factor in adapting to a variety of environments. Based on the previous studies, adaptive genetic variation in the glycerol kinase GlpK, the glycerol 3-phosphotransferase, contributes to the fitness of bacteria in human bodies, such as Mycobacterium tuberculosis and Escherichia coli. Thus, this study aimed to explore the molecular evolution and function of glpK in P. aeruginosa. Using extensive population genomic data, we have identified the prevalence of two glpK copies in P. aeruginosa that clustered into distinct branches, which were later known as Clade 1 and 2. The evolution analysis revealed that glpK in Clade 1 derived from an ancestral P. aeruginosa species and the other from an ancient horizontal gene transfer event. In addition, we confirmed that the GlpK in Clade 2 still retained glycerol kinase activity but was much weaker than that of GlpK in Clade 1. We demonstrated the importance of the critical amino acid Q70 in GlpK glycerol kinase activity by point mutation. Furthermore, Co-expression network analysis implied that the two glpK copies of P. aeruginosa regulate separate networks and may be a strategy to improve fitness in P. aeruginosa.
Subject(s)
Glycerol Kinase , Pseudomonas aeruginosa , Humans , Glycerol/metabolism , Glycerol Kinase/genetics , Glycerol Kinase/metabolism , Phosphorylation , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
BACKGROUND: Xp21 contiguous gene deletion syndrome is a rare genetic metabolic disorder with poor prognosis in infants, involving deletions of one or more genes in Xp21. When deletions of adrenal hypoplasia (AHC), Duchenne muscular dystrophy (DMD), and chronic granulomatosis (CGD) loci are included, complex glycerol kinase deficiency (CGKD) can be diagnosed. We present a case of CGKD that was initially misdiagnosed and died during treatment in our hospital in terms of improving our understanding of the clinical features and diagnosis of this disease, as well as highlighting the need for more precise dosing of corticosteroid replacement therapy. CASE PRESENTATION: A 48-day-old full-term male infant was transferred to our medical center with global growth delay and persistent vomiting. Routine laboratory tests revealed hyperkalemia, hyponatremia, and a high level of creatine kinase. The initial diagnosis was adrenal cortical hyperplasia (ACH), then revised to adrenocortical insufficiency with a normal level of ACTH detected. After supplementing the routine lipid test and urinary glycerol test, CGKD was diagnosed clinically due to positive triglyceridemia and urinary glycerol, and the follow-up gene screening further confirmed the diagnosis. The boy kept thriving after corticosteroid replacement and salt supplementation. While levels of serum ACTH and cortisol decreased and remained low after corticosteroid replacement was administered. The patient died of acute type 2 respiratory failure and hypoglycemia after an acute upper respiratory tract infection, which may be the result of adrenal crisis after infection. Infants with CGKD have a poor prognosis, so physicians should administer regular follow-ups, and parents counseling during treatment to improve the survival of patients. CONCLUSIONS: Overall, CGKD, although rare, cannot be easily excluded in children with persistent vomiting. Extensive blood tests can help to detect abnormal indicators. Adrenal crisis needs to be avoided as much as possible during corticosteroid replacement therapy.
Subject(s)
Adrenal Insufficiency , Glycerol Kinase , Adrenal Insufficiency/diagnosis , Adrenal Insufficiency/genetics , Adrenocorticotropic Hormone , Child , China , Delayed Diagnosis , Glycerol , Glycerol Kinase/genetics , Humans , Hypoadrenocorticism, Familial , Infant , Male , VomitingABSTRACT
Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.
Subject(s)
Bacillus subtilis , Glycerol Kinase , Chromosomes/metabolism , Escherichia coli/metabolism , Glycerol/metabolism , Glycerol Kinase/chemistry , Glycerol Kinase/genetics , Glycerol Kinase/metabolism , Mutagenesis, Site-DirectedABSTRACT
While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: ⢠Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. ⢠CbbX is essential for the efficiency of RuBisCO in C. necator H16. ⢠Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.
Subject(s)
Aquaporins , Cupriavidus necator , Escherichia coli Proteins , Aquaporins/metabolism , Carbon Dioxide/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Glycerol/metabolism , Glycerol Kinase/genetics , Glycerol Kinase/metabolism , Ribulose-Bisphosphate Carboxylase/metabolismABSTRACT
The fall armyworm (FAW), Spodoptera frugiperda, is native to the tropical and subtropical areas of the American continent and is one of the world's most destructive insect pests and invaded Africa and spread to most of Asia in two years. Glycerol is generally used as a cryoprotectant for overwintering insects in cold areas. In many studies, the increase in glycerol as a main rapid cold hardening (RCH) factor and enhancing the supercooling point was revealed at low temperatures. There are two genes, including glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), that were identified as being associated with the glycerol synthesis pathway. In this study, one GPDH and two GK sequences (GK1 and GK2) were extracted from FAW transcriptome analysis. RNA interference (RNAi) specific to GPDH or GK1 and GK2 exhibited a significant down-regulation at the mRNA level as well as a reduction in survival rate when the RNAi-treated of FAW larvae post a RCH treatment. Following a cold period, an increase in glycerol accumulation was detected utilizing high-pressure liquid chromatography and colorimetric analysis of glycerol quantity in RCH treated hemolymph of FAW larvae. This research suggests that GPDH and GK isozymes are linked to the production of a high quantity of glycerol as an RCH factor, and glycerol as main cryoprotectant plays an important role in survival throughout the cold period in this quarantine pest studied.
Subject(s)
Glycerol , Moths , Animals , Cryoprotective Agents , Glycerol/metabolism , Glycerol Kinase/genetics , Glycerolphosphate Dehydrogenase/metabolism , Larva/physiology , Moths/metabolism , Spodoptera/genetics , Spodoptera/metabolismABSTRACT
African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Glycerol Kinase/blood , Lysophospholipase/blood , Protozoan Proteins/blood , Serologic Tests/methods , Trypanosoma/immunology , Trypanosomiasis, Bovine/diagnosis , Animals , Cattle , Glycerol Kinase/genetics , Glycerol Kinase/immunology , Lysophospholipase/genetics , Lysophospholipase/immunology , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Trypanosoma/classification , Trypanosoma/enzymology , Trypanosoma/genetics , Trypanosomiasis, Bovine/blood , Trypanosomiasis, Bovine/parasitologyABSTRACT
Complex glycerol kinase deficiency (CGKD) is a rare genetic syndrome which belongs to the group of contiguous gene syndromes and is caused by microdeletion of genes located in Xp21. Patients with CGKD present with features characteristic for adrenal hypoplasia, glycerol kinase deficiency, Duchenne muscular dystrophy and sometimes intellectual disability. We present a long-term follow-up of two unrelated boys with molecular diagnosis of complex glycerol kinase deficiency. Genetic examinations in both patients revealed a deletion on Xp21 chromosome including complete deletion of NR0B1 and GK genes. Additionally in patient 2 IL1RAPL1 genes were deleted. In separate MLPA test DMD gene deletion was diagnosed in both patients as follow: in patient 1 whole gene while in patient 2 the C-terminal region of DMD was deleted. Although the first symptom in both was salt loss syndrome, the course of the disease was different for them. We share our experience resulting from the opportunity of caring for patients with this rare disease from the beginning of their life to the end of pediatric care.
Subject(s)
Glycerol Kinase , Child , Follow-Up Studies , Glycerol Kinase/genetics , Humans , Hypoadrenocorticism, Familial , Male , SyndromeABSTRACT
Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
Subject(s)
Adaptation, Physiological/genetics , Deuterium/metabolism , Escherichia coli K12/metabolism , Isotope Labeling/methods , Neutrons , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography/methods , Culture Media/chemistry , Culture Media/pharmacology , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli K12/drug effects , Escherichia coli K12/genetics , Escherichia coli K12/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Essential , Glycerol/metabolism , Glycerol/pharmacology , Glycerol Kinase/genetics , Glycerol Kinase/metabolism , Mutation , Neutron Diffraction , Potassium Channels/genetics , Potassium Channels/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Selection, Genetic , Sigma Factor/genetics , Sigma Factor/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Whole Genome SequencingABSTRACT
BACKGROUND: A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. RESULTS: We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. CONCLUSION: The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering , Recombinant Fusion Proteins/biosynthesis , Asparaginase/genetics , Asparaginase/metabolism , Down-Regulation , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Gene Knockout Techniques , Genes, Bacterial , Glycerol Kinase/genetics , Glycerol Kinase/metabolism , Glycerolphosphate Dehydrogenase/genetics , Glycerolphosphate Dehydrogenase/metabolism , Signal Transduction , Stress, Physiological , Up-Regulation , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/geneticsABSTRACT
Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.
Subject(s)
Aldehyde-Lyases/genetics , Biosynthetic Pathways/genetics , Dihydroxyacetone/biosynthesis , Escherichia coli K12/enzymology , Escherichia coli K12/genetics , Escherichia coli Proteins/genetics , Gene Expression , Genes, Bacterial , Glycerol/metabolism , Alleles , Fructosephosphates/metabolism , Gene Deletion , Glucose/metabolism , Glucosephosphate Dehydrogenase/genetics , Glycerol Kinase/genetics , Isoenzymes/genetics , Pentose Phosphate Pathway/genetics , Phosphofructokinases/chemistry , Phosphofructokinases/genetics , Sugar Alcohol Dehydrogenases/geneticsABSTRACT
BACKGROUND AND AIMS: Glycerol kinase deficiency (GKD) is a rare genetic disorder characterized by hyperglycerolemia and glyceroluria, which could be misdiagnosed as a moderate to severe hypertriglyceridemia (HTG). We aimed to describe four novel cases of GKD, to complete a systematic review of all cases of isolated GKD published so far, and to develop a suspicion clinical diagnostic score for GKD. METHODS: We reported four cases with suspicion of GKD and compared their phenotype with 584 males with triglycerides (TG) > 300 mg/dL, selected as control group (HTG non-GKD). The GK gene was sequenced in all cases. Lipoprotein particle concentrations were measured in all cases with GKD. The systematic review involved a PubMed, Cochrane and Scopus databases search to identify anthropometric and biochemical characteristics of all described cases with GKD. RESULTS: The systematic review retrieved a total of 15 articles involving 39 subjects with GKD. GKD cases reported a history of high TG levels resistant to lipid-lowering therapy. Compared to GKD subjects (n = 43), HTG non-GKD subjects (n = 584) showed significantly higher BMI, total cholesterol, non-HDL cholesterol and gamma-glutamyltransferase, significantly lower HDL cholesterol and TG, and higher prevalence of diabetes. The proposed diagnostic score was significantly higher in GKD than in HTG non-GKD subjects. CONCLUSIONS: This is the first systematic review that compiles all GKD cases reported to date including 4 novel cases, and examine the differential GKD phenotype compared to other types of HTG. The proposed score would have a broad utility in clinical practice to avoid unwarranted lipid lowering treatment in GKD patients.
Subject(s)
Glycerol Kinase , Hypertriglyceridemia , Adult , Glycerol Kinase/genetics , Humans , Hypertriglyceridemia/diagnosis , Lipoproteins , Male , Phenotype , TriglyceridesABSTRACT
We report a case of Xp21 deletion syndrome, a contiguous gene syndrome associating glycerol kinase deficiency, Duchenne muscular dystrophy, and congenital adrenal hypoplasia. This results in a contraindication to the use of all halogenated agents and of propofol. We used regional anesthesia combined with dexmedetomidine and ketamine. Previously, the patient had received inadvertently a propofol-based total intravenous anesthesia (TIVA) with no clinical side effects. We were unfortunately unable to document the metabolic consequences of this glycerol load. We suggest that if propofol is deemed necessary in such cases, it should only be used as a bolus dose of a 2% solution.