Inactivation Of Pyruvate Formate-lyase Research Articles

In silico profiling of cell growth and succinate production in Escherichia coli NZN111.

BackgroundSuccinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB, respectively. However, this double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and consequently disables proper growth under anaerobic conditions.ResultsIn this study, genome-scale metabolic core model of E. coli was reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strain E. coli NZN111. Non-optimized artificial centering hit-and-run (ACHR) method and metabolite flux-sum analysis were utilized to evaluate metabolic characteristics of strains. Thus, metabolic characteristics of the strains wild-type E. coli, ldhA mutant, pflB mutant, and NZN111 under anaerobic conditions were successfully unraveled.Conclusions We found a viewpoint contrary to the widespread realization that an NADH/NAD+ in NZN111 mainly resulted from the inactivation of PFL rather than the inactivation of LDH. In addition, the two alternative anaerobic fermentation pathways, lactate and ethanol production pathways, were blocked owing to the disruption of ldhA and pflB, resulting in insufficient NAD+ regeneration to oxidize or metabolize glucose for cell growth. Furthermore, we speculated reaction NADH16, the conversion of ubiquinone-8 (q8) to ubiquinol-8 (q8h2), as a potential amplification target for anaerobically improving cell growth and succinate production in NZN111.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0125-5) contains supplementary material, which is available to authorized users.

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1mol of CO2 was fixated in 1mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD+ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD+ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72h, 14.5gL−1 of glucose was consumed to generate 12.08gL−1 of succinic acid. Furthermore, under optimized condition of CO2 supply, the succinic acid productivity and the CO2 fixation rate reached 223.88mgL−1h−1 and 83.48mgL−1h−1, respectively.

Escherichia coli strains engineered for homofermentative production of D-lactic acid from glycerol.

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (DeltapflB) and fumarate reductase (DeltafrdA) (strain LA01) and (ii) inactivation of fumarate reductase (DeltafrdA), phosphate acetyltransferase (Deltapta), and alcohol/acetaldehyde dehydrogenase (DeltaadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Deltadld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Deltadld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.

Fermentation of Glycerol to Succinate by Metabolically Engineered Strains of Escherichia coli

The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD(+) ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.

Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition

The effects of several single-gene knockout mutants ( pykF, ppc, pflA, pta, and adhE mutants) on the metabolic flux distribution in Escherichia coli were investigated under microaerobic condition. The intracellular metabolite concentrations and enzyme activities were measured, and the metabolic flux distribution was computed to study the metabolic regulation in the cell. The pflA, pta and ppc mutants produced large amount of lactate when using glucose as a carbon source under microaerobic condition. Comparing the flux distribution and the enzyme activities in the mutants, it was shown that the lactate production was promoted by the inactivation of pyruvate formate lyase and the resulting overexpression of lactate dehydrogenase. The flux through Pta–Ack pathways and the ethanol production were limited by the available acetyl coenzyme A. It was shown that the glycolysis was activated in pykF mutant in microaerobic culture. The glycolytic flux was related with Pyk activity except for pykF mutant. The cell growth rate was shown to be affected by the flux through phosphoenolpyruvate carboxylase. The quantitative regulation analysis was made based on the deviation indexes.

Inhibitory effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid production in dental plaque in vivo.

This study was conducted to find out whether sorbitol inhibits the sugar metabolism of Streptococcus mutans in vitro and the acid production in dental plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol-containing medium. The rate of acid production from sugars was estimated with a pH stat. The rate of acid production from glucose or sucrose was not changed at various concentrations of oxygen. By the addition of sorbitol to sugar, however, the acid production was decreased with increasing levels of oxygen. Intracellular NADH/NAD+ ratio and (dihydroxyacetone-phosphate+glyceraldehyde-phosphate)/3-phosphoglycerate ratio were high whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited the acid production in dental plaque in vivo. These results suggest that the increased NADH/NAD+ ratio during sorbitol metabolism through the inactivation of pyruvate formate-lyase by oxygen inhibited glyceraldehyde-phosphate dehydrogenase and then the acid production of S. mutans and the one in dental plaque.

Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR.

Pyruvate formate-lyase from Escherichia coli (EC 2.3.1.54; PFL) catalyzes the reversible anaerobic conversion of pyruvate and CoA into acetyl-CoA and formate. Active PFL contains a novel alpha-carbon centered glycyl radical at G734 that is required for its catalytic activity. Two adjacent cysteine residues, C418 and C419, are essential for PFL activity according to site-directed mutagenesis studies. Upon exposure to air, active PFL loses its activity with the concomitant loss of the glycyl radical. Previous EPR studies of dioxygen inactivation of PFL revealed protein-based peroxyl and sulfinyl radicals during the manual mixing and quenching process [Reddy et al. (1998) Biochemistry 37, 558-563]. To probe the mechanism of this process, we carried out experiments using rapid freeze-quench EPR spectroscopy. Upon mixing of active wild type or C418A PFL with oxygenated solution, a short-lived radical intermediate appears at the earliest time point (10 ms), followed by the appearance of a long-lived sulfinyl radical. The axial EPR spectrum of this short-lived radical (g = 2.034, 2.007) is characteristic of a peroxyl radical. When C419A PFL or the double mutant [C418A/C419A] PFL was mixed with oxygenated solution, the peroxyl radical was also observed at 10 ms but in this case persisted over 12 s. These observations provide compelling evidence to support a proposed mechanism in which dioxygen quenches the glycyl radical in the active enzyme and the resulting peroxyl radical may react further with the sulfhydryl group of the C419 residue to form the sulfinyl radical.

Rate‐limiting steps of glucose and sorbitol metabolism in Streptococcus mutans cells exposed to air

It has been supposed that rate of sorbitol metabolism in the air-exposed streptococcal cells could be limited by the low capacity to regenerate nicotinamide adenine dinucleotide (NAD) from reduced NAD (NADH) following inactivation of pyruvate formate-lyase by oxygen. The rate-limiting steps, however, have not been identified. The aim of this study was to examine the effect of temporary exposure of the streptococcal cells to air on the intracellular flux of glucose and sorbitol metabolism by measuring acid excretion, fluorescence dependent on cellular level of NADH, glycolytic intermediates and enzyme activities. The exposure of cells to air decreased the acid excretions during glucose and sorbitol metabolism. The analysis of the glycolytic intermediates and the fluorescence suggested that the reduced level of acid excretion in the air-exposed glucose metabolizing cells resulted from the decrease in pyruvate catabolism. In the presence of sorbitol, the decreased acid production resulted from the reduced rates of the reactions catalyzed by sorbitol-phosphoenolpyruvate phosphotransferase and sorbitol 6-phosphate dehydrogenase because of shortage of substrates for these enzymes in addition to the decrease in pyruvate catabolism.