scholarly journals Microbial metabolism of C1 and C2 compounds. The involvement of glycollate in the metabolism of ethanol and of acetate by Pseudomonas AM1

1972 ◽  
Vol 128 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Patricia M. Dunstan ◽  
C. Anthony ◽  
W. T. Drabble
Keyword(s):  
Alcohol Dehydrogenase ◽  
Generation Time ◽  
Isocitrate Lyase ◽  
Microbial Metabolism ◽  
Malate Synthase ◽  
Short Term ◽  
Glyoxylate Bypass ◽  
Incubation Experiments

Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol. Although malate synthase is present in extracts of ethanol-grown Pseudomonas AM1, the activity of isocitrate lyase is negligible. Short-term incubation experiments with [14C]ethanol and [14C]acetate indicate that a novel pathway operates during growth of Pseudomonas AM1 on ethanol. Glycollate, glyoxylate and malate are probably intermediates in this pathway.

scholarly journals Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308

1987 ◽  
Vol 242 (3) ◽  
pp. 661-665 ◽  
Author(s):  
E M T el-Mansi ◽  
C MacKintosh ◽  
K Duncan ◽  
W H Holms ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activities ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Isocitrate Lyase ◽  
Malate Synthase ◽  
Structural Genes ◽  
Glyoxylate Bypass ◽  
Over Expression

A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed. This plasmid complements an aceA mutation. The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro. Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.

scholarly journals Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and β-hydroxybutyrate

1973 ◽  
Vol 132 (4) ◽  
pp. 797-801 ◽  
Author(s):  
Patricia M. Dunstan ◽  
C. Anthony
Keyword(s):  
Microbial Metabolism ◽  
The Novel ◽  
Short Term ◽  
Glyoxylate Bypass

Pseudomonas AM1 grows on β-hydroxybutyrate and methanol at similar rates. β-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C1 compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.

scholarly journals Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claudia Durall ◽  
Kateryna Kukil ◽  
Jeffrey A. Hawkes ◽  
Alessia Albergati ◽  
Peter Lindblad ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tca Cycle ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Control Strain ◽  
Genes Encoding ◽  
Pcc 6803

Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana

2001 ◽  
Vol 29 (2) ◽  
pp. 283-286 ◽  
Author(s):  
E. L. Rylott ◽  
M. A. Hooks ◽  
I. A. Graham
Keyword(s):  
Arabidopsis Thaliana ◽  
Enzyme Activities ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Molecular Genetic ◽  
Regulation Of Gene Expression ◽  
Growth Period ◽  
Malate Synthase ◽  
The Glyoxylate Cycle

Molecular genetic approaches in the model plant Arabidopsis thaliana (ColO) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: β-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolasemediated steps of β-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of β-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

scholarly journals RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

2004 ◽  
Vol 186 (9) ◽  
pp. 2798-2809 ◽  
Author(s):  
Robert Gerstmeir ◽  
Annette Cramer ◽  
Petra Dangel ◽  
Steffen Schaffer ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Isocitrate Lyase ◽  
Transcriptional Regulator ◽  
Regulatory Elements ◽  
Malate Synthase ◽  
Acetate Kinase ◽  
Acetate Metabolism ◽  
Peptide Mass ◽  
Specific Activities ◽  
High Level

ABSTRACT The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

scholarly journals The Identification and Characterization of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 523-530
Author(s):  
Aileen K W Taguchi ◽  
Elton T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Isocitrate Lyase ◽  
Carbon Sources ◽  
Recessive Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence ◽  
Adh Activity ◽  
Identification And Characterization

ABSTRACT The alcohol dehydrogenase II isozyme (enzyme, ADHII; structural gene, ADH2) of the yeast, Saccharomyces cerevisiae, is under stringent carbon catabolite control. This cytoplasmic isozyme exhibits negligible activity during growth in media containing fermentable carbon sources such as glucose and is maximal during growth on nonfermentable carbon sources. A recessive mutation, adr6-1, and possibly two other alleles at this locus, were selected for their ability to decrease Ty-activated ADH2-6 c expression. The adr6-1 mutation led to decreased ADHII activity in both ADH2-6c and ADH2+ strains, and to decreased levels of ADH2 mRNA. Ty transcription and the expression of two other carbon catabolite regulated enzymes, isocitrate lyase and malate dehydrogenase, were unaffected by the adr6-1 mutation. adr6-1/adr6-1strains were defective for sporulation, indicating that adr6 mutations may have pleiotropic effects. The sporulation defect was not a consequence of decreased ADH activity. Since the ADH2-6c mutation is due to insertion of a 5.6-kb Ty element at the TATAA box, it appears that the ADR6+-dependent ADHII activity required ADH2 sequences 3′ to or including the TATAA box. The ADH2 upstream activating sequence (UAS) was probably not required. The ADR6 locus was unlinked to the ADR1 gene which encodes another trans-acting element required for ADH2 expression.

scholarly journals The carbonate concentration mechanism of Pyropia yezoensis (Rhodophyta): evidence from transcriptomics and biochemical data

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Baoyu Zhang ◽  
Xiujun Xie ◽  
Xuehua Liu ◽  
Linwen He ◽  
Yuanyuan Sun ◽  
...  
Keyword(s):  
Photosynthetic Efficiency ◽  
Inorganic Carbon ◽  
De Novo ◽  
Isocitrate Lyase ◽  
Life Cycles ◽  
Malate Synthase ◽  
Pyropia Yezoensis ◽  
Cdna Libraries ◽  
Life Stages ◽  
Low Carbon

Abstract Background Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. Results Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. Conclusion We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

Increase in glyoxylate shunt enzymes during cellular morphogenesis in Myxococcus xanthus

1971 ◽  
Vol 17 (2) ◽  
pp. 209-211 ◽  
Author(s):  
J. Bland ◽  
Wu-Kuang Yeh ◽  
D. White ◽  
A. Hendricks
Keyword(s):  
Actinomycin D ◽  
Isocitrate Lyase ◽  
Myxococcus Xanthus ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Cellular Morphogenesis

Isocitrate lyase (EC. 4.1.3.1) and malate synthase (EC. 4.1.3.2) increase during microcyst formation in Myxococcus xanthus. The increase in activity is inhibited by chloramphenicol and actinomycin-D.

Regulation of Peroxidase Transcript Levels in Liquid Cultures of the Ligninolytic Fungus Pleurotus eryngii

1999 ◽  
Vol 65 (10) ◽  
pp. 4458-4463 ◽  
Author(s):  
Francisco Javier Ruiz-Dueñas ◽  
Francisco Guillén ◽  
Susana Camarero ◽  
Marta Pérez-Boada ◽  
María Jesús Martínez ◽  
...  
Keyword(s):  
Northern Blotting ◽  
Pleurotus Eryngii ◽  
Versatile Peroxidase ◽  
Oxygen Species ◽  
Short Term ◽  
Transcript Levels ◽  
Liquid Cultures ◽  
Incubation Experiments ◽  
Peptone Medium ◽  
First Time

ABSTRACT A versatile peroxidase able to oxidize Mn2+ as well as phenolic and nonphenolic aromatic compounds is produced in peptone-containing liquid cultures of Pleurotus eryngiiencoded by the gene mnpl. The regulation of its transcript levels was investigated by Northern blotting of total RNA. High-peroxidase transcripts and activity were found in cultures grown in glucose-peptone medium, whereas only basal levels were detected in glucose-ammonium medium. The addition of more than 25 μM Mn2+ to the former medium did not result in detectable peroxidase transcripts or activity. Potential regulators were also added to isolated mycelium. In this way, it was shown that high transcript levels (in peroxidase-expressing mycelium) were maintained on peptone, whereas expression was not induced in short-term incubation experiments. Similar results were obtained with Mn2+ ions. Strong induction of mnpl expression was caused by exogenous H2O2 or by continuous H2O2 generation during redox cycling of menadione. By the use of the latter system in the presence of Fe3+, which catalyzes the reduction of H2O2 to hydroxyl radical, it was shown for the first time that the presence of this strong oxidant causes a rapid increase of the transcripts of a ligninolytic peroxidase. In conclusion, peptone and Mn2+ affect the levels of transcripts of this versatile peroxidase in culture, and reduced oxygen species induce short-term expression in isolated mycelium, probably via a stress response mechanism.

Incomplete Glyoxysomes Appearing at a Late Stage of Maturation of Cucumber Seeds

1979 ◽  
Vol 34 (12) ◽  
pp. 1232-1236 ◽  
Author(s):  
Wolfram Koller ◽  
Jürgen Frevert ◽  
Helmut Kindi
Keyword(s):  
Late Stage ◽  
Citrate Synthase ◽  
De Novo ◽  
Density Gradient Centrifugation ◽  
Isocitrate Lyase ◽  
Gradient Centrifugation ◽  
Protein Body ◽  
Electrophoretic Analysis ◽  
Malate Synthase ◽  
Lyase Activity

Seeds of cucumber fruits at a late stage of ripening were analyzed for microbodies and micro­body components. On isopycnic density gradient centrifugation of homogenates in the presence of EDTA, several particulate fractions were obtained: a light membraneous fraction (density d = 1.09-1.11 kg × 1-1), a mitochondria-enriched fraction (d = 1.21 kg × 1-1), a microbody-enriched fraction (d = 1.23 kg × 1-1), and a protein body fraction (d= 1.26 - 1.29 kg × 1-1). Microbo­dies were revealed by exactly coinciding peaks of malate synthase, catalase and crotonase; small proportions of citrate synthase and malate dehydrogenase were also present in this zone. Isocitrate lyase activity, however, did not occur in the seeds at this stage. The examination of enzyme activi­ties indicated the presence of microbodies which cannot function as competent glyoxysomes be­cause of the lack of isocitrate lyase. Moreover, de novo synthesis from [3H] leucine could be de­monstrated for malate synthase by means of immunoprecipitation of newly synthesized malate synthase and subsequent electrophoretic analysis.

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