Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype

ABSTRACT We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.

Download Full-text

Effects of Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by Metabolically Engineered Strains of Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.1715-1727.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 1715-1727 ◽

Cited By ~ 181

Author(s):

G. N. Vemuri ◽

M. A. Eiteman ◽

E. Altman

Keyword(s):

Escherichia Coli ◽

Succinic Acid ◽

Pyruvate Carboxylase ◽

Isocitrate Lyase ◽

Tricarboxylic Acid Cycle ◽

Metabolic Flexibility ◽

Dual Phase ◽

Anaerobic Conditions ◽

Carboxylase Activity ◽

Lyase Activity

ABSTRACT Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

Download Full-text

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

Biochemical Society Transactions ◽

10.1042/bst0290283 ◽

2001 ◽

Vol 29 (2) ◽

pp. 283-286 ◽

Cited By ~ 54

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.

Download Full-text

Isocitrate lyase fromAspergillus nidulans: crystallization and X-ray analysis of a glyoxylate cycle enzyme

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444997002680 ◽

1997 ◽

Vol 53 (4) ◽

pp. 488-490 ◽

Cited By ~ 1

Author(s):

S. J. Langridge ◽

P. J. Baker ◽

J. R. De Lucas ◽

S. E. Sedelnikova ◽

G. Turner ◽

...

Keyword(s):

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

X Ray ◽

Cycle Enzyme

Download Full-text

Molecular analysis of the Penicillium marneffei glyceraldehyde-3-phosphate dehydrogenase-encoding gene (gpdA) and differential expression of gpdA and the isocitrate lyase-encoding gene (acuD) upon internalization by murine macrophages

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/002832-0 ◽

2008 ◽

Vol 57 (11) ◽

pp. 1322-1328 ◽

Cited By ~ 15

Author(s):

Sophit Thirach ◽

Chester R. Cooper ◽

Nongnuch Vanittanakom

Keyword(s):

Differential Expression ◽

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

Evolutionary Relationship ◽

Amino Acid Sequences ◽

Penicillium Marneffei ◽

Macrophage Infection ◽

Dimorphic Fungus ◽

Isolation And Characterization ◽

Encoding Gene

Penicillium marneffei is an intracellular dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients. The factors that affect the pathogenicity of this fungus remain unclear. Here, we report the isolation and characterization of the gpdA cDNA and genomic clones encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in P. marneffei. Phylogenetic analysis of GAPDH amino acid sequences demonstrated the evolutionary relationship of P. marneffei to other fungi, including the intracellular pathogen Ajellomyces capsulatus. To assess the central importance of phagocytic cells in defence against P. marneffei infection, we used Northern blotting to investigate the response of the isocitrate lyase-encoding gene (acuD) and gpdA to nutrient deprivation inside macrophages. The results revealed that after macrophage internalization, the gene involved in the glyoxylate cycle, acuD, showed higher expression levels as early as 2 h from the start of co-incubation, and the differential expression could be observed again at 8 h after infection. In contrast, the expression of gpdA was downregulated in the yeast phase, as well as during macrophage infection after 2, 4 and 8 h of infection. The induction of P. marneffei acuD was shown to be coordinated with the downregulation of the glycolytic gpdA gene, implying that the cytoplasmic environment of macrophages is deficient in glucose and the glyoxylate pathway could be used by this pathogen to allow subsistence on two-carbon compounds within the host cell following its intracellular persistence.

Download Full-text

Whole-Genome Transcriptional Profiling of Bradyrhizobium japonicum during Chemoautotrophic Growth

Journal of Bacteriology ◽

10.1128/jb.00543-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6697-6705 ◽

Cited By ~ 24

Author(s):

William L. Franck ◽

Woo-Suk Chang ◽

Jing Qiu ◽

Masayuki Sugawara ◽

Michael J. Sadowsky ◽

...

Keyword(s):

Bradyrhizobium Japonicum ◽

Carbon Fixation ◽

Cultured Cells ◽

Isocitrate Lyase ◽

Nitrogenase Activity ◽

Glyoxylate Cycle ◽

Transcriptional Profiling ◽

Pentose Phosphate ◽

Hydrogen Gas ◽

Essential Components

ABSTRACT Bradyrhizobium japonicum is a facultative chemoautotroph capable of utilizing hydrogen gas as an electron donor in a respiratory chain terminated by oxygen to provide energy for cellular processes and carbon dioxide assimilation via a reductive pentose phosphate pathway. A transcriptomic analysis of B. japonicum cultured chemoautotrophically identified 1,485 transcripts, representing 17.5% of the genome, as differentially expressed when compared to heterotrophic cultures. Genetic determinants required for hydrogen utilization and carbon fixation, including the uptake hydrogenase system and components of the Calvin-Benson-Bassham cycle, were strongly induced in chemoautotrophically cultured cells. A putative isocitrate lyase (aceA; blr2455) was among the most strongly upregulated genes, suggesting a role for the glyoxylate cycle during chemoautotrophic growth. Addition of arabinose to chemoautotrophic cultures of B. japonicum did not significantly alter transcript profiles. Furthermore, a subset of nitrogen fixation genes was moderately induced during chemoautotrophic growth. In order to specifically address the role of isocitrate lyase and nitrogenase in chemoautotrophic growth, we cultured aceA, nifD, and nifH mutants under chemoautotrophic conditions. Growth of each mutant was similar to that of the wild type, indicating that the glyoxylate bypass and nitrogenase activity are not essential components of chemoautotrophy in B. japonicum.

Download Full-text

THIAMINE-RESPONSIVE LACTIC ACIDOSIS IN A PATIENT WITH DEFICIENT LOW-KM PYRUVATE CARBOXYLASE ACTIVITY IN LIVER

PEDIATRICS ◽

10.1542/peds.50.5.702 ◽

1972 ◽

Vol 50 (5) ◽

pp. 702-711

Author(s):

Michèle G. Brunette ◽

Edgard Delvin ◽

Bernard Hazel ◽

Charles R. Scriver

Keyword(s):

Lactic Acidosis ◽

Pyruvate Dehydrogenase ◽

Pyruvate Carboxylase ◽

Acid Metabolism ◽

Psychomotor Retardation ◽

Dietary Control ◽

Carboxylase Activity ◽

Cultured Skin ◽

Carbohydrate Feeding ◽

Partial Deficiency

The cause of severe intermittent lactic acidosis was investigated in a female infant with profound psychomotor retardation. Hypoglycemia, hyperpyruvic acidemia, and hyperalaninemia were identified in the newborn period. A triad of lactate, pyruvate, and alanine accumulation persisted throughout infancy, and ACTH, anorexia, and high carbohydrate feeding further provoked their accumulation. Careful dietary control or thiamine-HCl supplementation (5 to 20 mg/day) ameliorated the metabolic abnormality. Pyruvate dehydrogenase activity (which is thiamine-dependent) was normal in leukocytes and cultured skin fibroblasts. Hepatic pyruvate carboxylase activity (which is biotin-dependent) was found to comprise more than one component. There was a partial deficiency of total hepatic pyruvate carboxylase activity in the patient. The loss of activity was confined to the low-Km component of the enzyme which serves pvruvate metabolism in the physiological range. A defect in glucogenesis causing hypoglycemia, pyruvate accumulation with lactic acidosis, and aberrant amino acid metabolism can be attributed to the abnormality of pyruvate carboxylase. The response to thiamine in our patients may reflect activation of a normal "shunt" mechanism for pyruvate disposal via pyruvate dehydrogenase.

Download Full-text

Role of salicylic acid in regulation of cadmium toxicity in wheat ( Triticum aestivum L.)

Acta Agronomica Hungarica ◽

10.1556/aagr.57.2009.3.7 ◽

2009 ◽

Vol 57 (3) ◽

pp. 321-333 ◽

Cited By ~ 1

Author(s):

H. Moussa ◽

S. EL-Gamal

Keyword(s):

Pyruvate Carboxylase ◽

Cadmium Toxicity ◽

Triticum Aestivum L ◽

Carboxylase Activity ◽

Bisphosphate Carboxylase ◽

Cd Accumulation ◽

Phosphoenol Pyruvate ◽

Relative Water ◽

Aba Content

Treatment with CdCl 2 (0, 100, 400 and 1000 μM) resulted in the inhibition of root dry biomass and root elongation and to increased Cd accumulation in the roots. These treatments also decreased the relative water content, chlorophyll content, 14 CO fixation, phosphoenol pyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase activity and abscisic acid (ABA) content, while increasing the malondialdehyde, hydrogen peroxide and free proline contents and causing changes in the chloroplast and root ultrastructure. Pretreatment of seeds with SA (500 μM) for 20 h resulted in the amelioration of these effects.

Download Full-text

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Planta ◽

10.1007/bf00388635 ◽

1978 ◽

Vol 139 (3) ◽

pp. 239-243 ◽

Cited By ~ 14

Author(s):

M. Perl

Keyword(s):

Pyruvate Carboxylase ◽

Seedling Development ◽

Carboxylase Activity ◽

Phosphoenol Pyruvate Carboxylase ◽

Phosphoenol Pyruvate

Download Full-text

Acetate metabolism in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m78-027 ◽

1978 ◽

Vol 24 (2) ◽

pp. 149-153 ◽

Cited By ~ 5

Author(s):

T. M. Lakshmi ◽

Robert B. Helling

Keyword(s):

Isocitrate Dehydrogenase ◽

Citrate Synthase ◽

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

Acetate Metabolism ◽

Wild Type ◽

Intermediary Metabolites ◽

Lyase Activity ◽

Acetate Medium ◽

The Glyoxylate Cycle

Levels of several intermediary metabolites were measured in cells grown in acetate medium in order to test the hypothesis that the glyoxylate cycle is repressed by phosphoenolpyruvate (PEP). Wild-type cells had less PEP than either isocitrate dehydrogenase – deficient cells (which had greater isocitrate lyase activity than the wild type) or isocitrate dehydrogenase – deficient, citrate synthase – deficient cells (which are poorly inducible). Thus induction of the glyoxylate cycle is more complicated than a simple function of PEP concentration. No correlation between enzyme activity and the level of oxaloacetate, pyruvate, or citrate was found either. Citrate was synthesized in citrate synthase – deficient mutants, possibly via citrate lyase.

Download Full-text