Enzymes Of Central Carbon Metabolism Research Articles

Relationship between glucose catabolism and xanthan production in Xanthomonas oryzae pv. oryzae

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.

Gene Organization and CO<sub>2</sub>-Responsive Expression of Four <i>cbb</i> Operons in the Biomining Bacterium <i>Acidithiobacillus Ferrooxidans</i>

Acidithiobacillus ferrooxidans is an obligately chemolithoautotrophic, -proteobacterium that fixes CO2 by the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle. Our objective is to identify genes potentially involved in CO2 fixation and to advance our understanding of how they might be regulated in response to environmental signals. Bioinformatic analyses, based on the complete genome sequence of the type strain ATCC 23270, identified five cbb gene clusters four of which we show experimentally to be operons. These operons are predicted to encode: (i) the components of the carboxysome and one copy of form I RubisCO (cbb1 operon), (ii) a second copy of form I RubisCO (cbb2 operon), (iii) enzymes of central carbon metabolism (cbb3 operon), (iv) a phosphoribulokinase and enzymes of sulfur metabolism (cbb4 operon) and RubisCO form II (cbb5 gene cluster). In addition, the gene for a LysR-type transcriptional regulator CbbR was identified immediately upstream and in divergent orientation to the cbb1 operon and another associated with the cbb5 gene cluster. A. ferrooxidans was grown under different concentrations of CO2 (2.5 to 20% [v/v]), and levels of mRNA and protein were evaluated by qPCR and Western blotting, respectively. CbbR binding to predicted promoter regions of operons cbb1-4 was assayed by EMSA This information permitted the formulation of models explaining how these operons might be regulated by environmental CO2 concentrations. These models were evaluated in vivo in a heterologous host, using cloned A. ferrooxidans cbbR to complement a mutant of the facultative chemoautotroph Ralstonia eutropha H16 lacking a functional cbbR. Cloned copies of A. ferrooxidans promoter regions were also introduced into R. eutropha to evaluate their ability to drive reporter gene expression. This work lays the framework for further studies that should result in a more comprehensive picture of how CO2 fixation is regulated in A. ferrooxidans.

Changes in the metabolome of associated with evolution in aerobic glucose-limited chemostats

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h(-1). It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5-10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5-10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.

Autotrophic CO2 fixation pathways in archaea (Crenarchaeota).

Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways. Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle. This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus. Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water. Yet, phosphoribulokinase activity was not detectable. Operation of the Calvin cycle remains to be demonstrated. Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue. Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp. strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle. This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus. Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail. Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation. However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration. We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways. The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

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.