Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.1 (pyruvate carboxylase)
1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) as anaplerotic enzymes for growth on carbohydrates. To analyze the significance of PCx for the amino acid production by this organism, the wild-type pyc gene, encoding PCx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of C. glutamicum were tested in comparison to the respective host strains. No PCx activity was observed in the pyc-inactive mutants whereas the pyc-overexpressing strains showed eight-to elevenfold higher specific PCx activity when compared to the host strains. In a detergent-dependent glutamate production assay, the pyc-overexpressing strain showed more than sevenfold higher, the PCx-deficient strain about twofold lower glutamate production than the wild-type. Overexpression of the pyc gene and thus increasing the PCx activity in a lysine-producing strain of C. glutamicum resulted in approximately 50% higher lysine accumulation in the culture supernatant whereas inactivation of the pyc gene led to a decrease by 60%. In a threonine-producing strain of C. glutamicum, the overexpression of the pyc gene led to an only 10 to 20% increase in threonine production, however, to a more than 150% increase in the production of the threonine precursor homoserine. These results identify the anaplerotic PCx reaction as a major bottleneck for amino acid production by C. glutamicum and show that the enzyme is an important target for the molecular breeding of hyperproducing strains.
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PMID:Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. 1132 86

Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.
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PMID:A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. 1187 15

Defects of lysine metabolism are rare, but hyperlysinemia is a concomitant of many inborn errors of metabolism, including urea cycle abnormalities, pyruvate carboxylase deficiency and L-2-hydroxyglutaric aciduria. We have hypothesized that mitochondrial lysine degradation is regulated by bioavailability of 2-oxoglutarate in the same compartment, and our studies in physiologic fluid derived from patients with the above described disorders supports our hypothsis. Our data further suggest that patients with isolated L-2-hydroxyglutaric aciduria may have a defect in 2-ketoglutarate metabolism. The current report summarizes our studies.
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PMID:Plasma lysine concentration and availability of 2-ketoglutarate in liver mitochondria. 1199 75

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.
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PMID:Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum. 1240 33

L-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years. Nowadays production exceeds 600,000 tons per year. Based on conventionally bred strains, further improvement of lysine productivity has been achieved by genetic engineering. Pyruvate carboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase and the specific lysine exporter were shown to be key enzymes for lysine production and were characterized in detail. Their combined engineering led to a striking increase in lysine formation. Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinated flux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cycling between C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle. Process economics have been optimized by developing repeated fed-batch techniques and technical continuous fermentations. In addition, on-line metabolic pathway analysis or flow cytometry may help to improve the fermentation performance. Finally, the availability of the Corynebacterium glutamicum genome sequence has a major impact on the improvement of the biotechnological manufacture of lysine. In this context, all genes of the carbon flow from sugar uptake to lysine secretion have been identified and are accessible to manipulation. The whole sequence information gives access to post genome technologies such as transcriptome analysis, investigation of the proteome and the active metabolic network. These multi-parallel working technologies will accelerate the generation of knowledge. For the first time there is a chance of understanding the overall picture of the physiological state of lysine overproduction in a technical environment.
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PMID:Biotechnological manufacture of lysine. 1252 89

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.
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PMID:Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression. 1274 42

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min(-1) (mg of protein)(-1)], threefold-increased pyruvate carboxylase activity [535 nmol min(-1) (mg of protein)(-1)], and elevated citrate synthase (CS) activity [292 nmol min(-1) (mg of protein)(-1)] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50 degrees C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity.
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PMID:Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants. 1283 72

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.
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PMID:Serial flux mapping of Corynebacterium glutamicum during fed-batch L-lysine production using the sensor reactor approach. 1476 Jun 90

Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate to yield propionyl-CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer of beta(8)alpha(8) barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S-based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites.
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PMID:Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit. 1532 73

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway.
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PMID:Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum. 1733 67


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