Metabolic Analysis of Wild-type Escherichia coli and a Pyruvate Dehydrogenase Complex (PDHC)-deficient Derivative Reveals the Role of PDHC in the Fermentative Metabolism of Glucose (original) (raw)
Related papers
Microbiology, 2010
The fermentative metabolism of D-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDHdeficient strain (Pdh") exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH-and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh" were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD + ratio supported the proposed role of PDH. Moreover, the NADH : NAD + ratio in a strain deficient in both PDH and the ox-PPP (Pdh"Zwf") was even lower than that observed for Pdh". Strain Pdh"Zwf" also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh". Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh" mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD + ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.
Applied and Environmental Microbiology, 2010
During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed b...
THE PLANT CELL ONLINE, 2012
Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H 2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H 2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.
Journal of Bacteriology, 2008
Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mu...
Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405
Indian Journal of Microbiology, 2008
Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H 2 and CO 2 . Very little is known about the factors that determine metabolic fl uxes infl uencing H 2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identifi ed genes encoding key enzymes in pyruvate catabolism and H 2 -synthesis pathways, and have confi rmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as "Energy-converting Hydrogenases", or via NAD(P)Hdependent Fe-only hydrogenases which would permit H 2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our fi ndings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H 2 synthesis is the fi rst step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confi rmed using both expression analysis and protein characterization.
Journal of Bacteriology, 2004
The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized. Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter. poxB mRNA expression was strongly induced by aeration and was repressed by glucose. Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted. Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region. The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production. Constitutive overproduction of PoxB in L. plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions. The ⌬poxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose). In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production. In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.
Biotechnology for biofuels, 2016
Fermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8*) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8*_ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol(-1) glucose), reduced the co-production yield. In this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9*_ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA (+)). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectiv...
The biochemistry of the pyruvate dehydrogenase complex
Biochemistry and Molecular Biology Education, 2003
In the Westernized world the daily dietary caloric requirements are roughly provided as follows: 40% from carbohydrates, 40% from fats, and 20% from proteins. In some populations in developing countries the daily caloric contribution from carbohydrate is even higher due to its readily available sources and relatively low cost. Glucose, the principal product of carbohydrate digestion, passes through a series of enzymatic steps first in the non-oxidative glycolytic pathway (from glucose to pyruvate) followed by efficient oxidative metabolism via the tricarboxylic acid cycle (from acetyl-CoA to CO 2 and H 2 O) to harness a portion of its potential energy as ATP. Interestingly, these two major pathways are directly linked by the pyruvate dehydrogenase complex (PDC) 1 localized in the mitochondrial matrix ). In the fed state acetyl-CoA generated from pyruvate (derived mostly from glucose and some dietary amino acids) is also utilized for the biosynthesis of lipids such as long chain fatty acids and cholesterol by lipogenic tissues (such as liver and adipose tissues and under special conditions in mammary glands during lactation and in the brain during the prenatal and early postnatal development). Additionally, amino acids from excess dietary protein are metabolized by several specialized reactions or pathways generating intermediates that have to be ultimately converted to pyruvate first and then to acetyl-CoA via PDC either for complete oxidation to CO 2 and H 2 O or for lipogenesis. PDC is the only known reaction in most eukaryotes to generate acetyl-CoA (two-carbon compound) from pyruvate (three-carbon compound). Since this is a physiologically irreversible reaction and since there is no other known reaction or pathway to convert the two-carbon compound to pyruvate (or its equivalent) for the synthesis of glucose in animals, the flux through PDC is tightly regulated to meet the specific metabolic and energetic needs of different tissues during the fed and fasting (starvation) states. This is accomplished by covalent modification of the ratelimiting component of the complex involving sophisticated interplay among the components of the complex and allosteric modulations by acetyl-CoA and NADH, the products of the reaction (and also of fatty acid oxidation). It is evident from these simple considerations that PDC plays a key role as a gatekeeper of both caloric and glucose homeostasis in mammals. In this review, we will discuss recent developments concerning the structure-function relationship of this multienzyme complex from various organisms with emphasis on regulatory aspects of the mammalian complex. Detailed accounts of various aspects of this complex can be found in several excellent reviews .
Microbial Physiology, 2008
The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS–). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS–Glc+), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (μ) higher than PB12 (0.21 and 0.13 h–1, respectively) while JM101 had a μ of 0.28 h–1. To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS– strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate ...