Succinate Secretion Research Articles

Substitute Yeast Extract While Maintaining Performance: Showcase Amorpha-4,11-Diene Production.

Yeast extract (YE) is a complex nutritional source associated with high performance on microbial production processes. However, its inherent compositional variability challenges its scalability. While prior efforts have focused on growth-associated products, the dynamics of growth-uncoupled production, which leads to higher production rates and conversion yields, still need to be explored. This production scenario is common in large-scale applications. This study presents a systematic approach to replace YE for the production of the terpene amorpha-4,11-diene in Escherichia coli. Sequential processing was successfully applied to identify glutamic acid, alanine, leucine, valine, isoleucine and glycine as the key amino acids (AAs) under slow-growth conditions. Thoroughly applying biomass retention as part of sequential processing increased production capacity by 45% using these AAs instead of YE. Further studies, including flux balance analyses, targeted pyruvate as the common AA precursor. The optimized fed-batch process feeding pyruvate with 0.09 gPyr h-1 enhanced amorpha-4,11-diene production by 37%, although adding only 1% carbon via pyruvate. Flux balance analysis revealed the criteria for optimum pyruvate feeding, for example, to prevent succinate secretion and maintain the NADH/NAD+ balance. These findings illustrate the interplay between media composition and metabolic activity and provide a successful guideline for identifying lean, best-performing media for industrial applications.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M.tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M.tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M.tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

ABSTRACT Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn’s disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1 α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate’s effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

Extracellular Succinate: A Physiological Messenger and a Pathological Trigger.

When tissues are under physiological stresses, such as vigorous exercise and cold exposure, skeletal muscle cells secrete succinate into the extracellular space for adaptation and survival. By contrast, environmental toxins and injurious agents induce cellular secretion of succinate to damage tissues, trigger inflammation, and induce tissue fibrosis. Extracellular succinate induces cellular changes and tissue adaptation or damage by ligating cell surface succinate receptor-1 (SUCNR-1) and activating downstream signaling pathways and transcriptional programs. Since SUCNR-1 mediates not only pathological processes but also physiological functions, targeting it for drug development is hampered by incomplete knowledge about the characteristics of its physiological vs. pathological actions. This review summarizes the current status of extracellular succinate in health and disease and discusses the underlying mechanisms and therapeutic implications.

Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit Tcell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and Tcells, we establish an invitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8+ Tcell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic Tcells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic Tcells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8+ Tcells to retain cytotoxicity and overcome a lactate-enriched TME.

Interplay between central carbon metabolism and metal homeostasis in mycobacteria and other human pathogens.

Bacterial nutrition is a fundamental aspect of pathogenesis. While the host environment is in principle nutrient-rich, hosts have evolved strategies to interfere with nutrient acquisition by pathogens. In turn, pathogens have developed mechanisms to circumvent these restrictions. Changing the availability of bioavailable metal ions is a common strategy used by hosts to limit bacterial replication. Macrophages and neutrophils withhold iron, manganese, and zinc ions to starve bacteria. Alternatively, they can release manganese, zinc, and copper ions to intoxicate microorganisms. Metals are essential micronutrients and participate in catalysis, macromolecular structure, and signalling. This review summarises our current understanding of how central carbon metabolism in pathogens adapts to local fluctuations in free metal ion concentrations. We focus on the transcriptomics and proteomics data produced in studies of the iron-sparing response in Mycobacterium tuberculosis, the etiological agent of tuberculosis, and consequently generate a hypothetical model linking trehalose accumulation, succinate secretion and substrate-level phosphorylation in iron-starved M. tuberculosis. This review also aims to highlight a large gap in our knowledge of pathogen physiology: the interplay between metal homeostasis and central carbon metabolism, two cellular processes which are usually studied separately. Integrating metabolism and metal biology would allow the discovery of new weaknesses in bacterial physiology, leading to the development of novel and improved antibacterial therapies.

Tumor Cells Dictate Anti-Tumor Immune Responses by Altering Pyruvate Utilization and Succinate Signaling in Cd8 <sup>+</sup> T Cells

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit CD8+ T cell metabolism and cytotoxic activity. It has been a challenge to dissect the metabolic interplay between tumors and T cells. Consequently, the mechanisms whereby the TME regulates CD8+ T cell function are not completely understood. Here we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as a direct inhibitor of CD8+ T cell cytotoxicity, revealing an unexpected metabolic shunt in the tricarboxylic acid (TCA) cycle in CD8+ T cells. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1), a G-protein coupled cell surface receptor. This leaves CD8+ T cells reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates (anaplerosis). In contrast, lactate reduces PC-mediated anaplerosis, thus depleting the TCA cycle and reducing SUCNR1 signaling. Notably, inhibition of pyruvate dehydrogenase (PDH) is sufficient to override lactate-mediated reprogramming, and to restore PC activity, succinate secretion, and autocrine activation of SUCNR1 both in vitro and in vivo using syngeneic tumor models. Thus, dissecting the CD8+ T cell-specific metabolic adaptation to the TME unexpectedly revealed that a TCA cycle succinate shunt and plasticity in pyruvate flux is a crucial determinant of CD8+ T cell anti-tumor activity. Furthermore, we identify PDH as a potential drug target to allow CD8+ T cells to retain cytotoxicity and overcome a lactate-rich TME. These studies demonstrate that the TME remodels CD8+ T cell pyruvate metabolism, leading to a reduction in CD8+ T cell cytotoxicity and anti-tumor immunity.

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte’s death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A 1H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

PH-Gated Succinate Secretion Regulates Muscle Remodeling in Response to Exercise

In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling upon exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho–cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner–Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus’ metabolism for future lignin valorization.

Updated and standardized genome-scale reconstruction of Mycobacterium tuberculosis H37Rv, iEK1011, simulates flux states indicative of physiological conditions

BackgroundThe efficacy of antibiotics against M. tuberculosis has been shown to be influenced by experimental media conditions. Investigations of M. tuberculosis growth in physiological conditions have described an environment that is different from common in vitro media. Thus, elucidating the interplay between available nutrient sources and antibiotic efficacy has clear medical relevance. While genome-scale reconstructions of M. tuberculosis have enabled the ability to interrogate media differences for the past 10 years, recent reconstructions have diverged from each other without standardization. A unified reconstruction of M. tuberculosis H37Rv would elucidate the impact of different nutrient conditions on antibiotic efficacy and provide new insights for therapeutic intervention.ResultsWe present a new genome-scale model of M. tuberculosis H37Rv, named iEK1011, that unifies and updates previous M. tuberculosis H37Rv genome-scale reconstructions. We functionally assess iEK1011 against previous models and show that the model increases correct gene essentiality predictions on two different experimental datasets by 6% (53% to 60%) and 18% (60% to 71%), respectively. We compared simulations between in vitro and approximated in vivo media conditions to examine the predictive capabilities of iEK1011. The simulated differences recapitulated literature defined characteristics in the rewiring of TCA metabolism including succinate secretion, gluconeogenesis, and activation of both the glyoxylate shunt and the methylcitrate cycle. To assist efforts to elucidate mechanisms of antibiotic resistance development, we curated 16 metabolic genes related to antimicrobial resistance and approximated evolutionary drivers of resistance. Comparing simulations of these antibiotic resistance features between in vivo and in vitro media highlighted condition-dependent differences that may influence the efficacy of antibiotics.ConclusionsiEK1011 provides a computational knowledge base for exploring the impact of different environmental conditions on the metabolic state of M. tuberculosis H37Rv. As more experimental data and knowledge of M. tuberculosis H37Rv become available, a unified and standardized M. tuberculosis model will prove to be a valuable resource to the research community studying the systems biology of M. tuberculosis.

Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni.

C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4–dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4–dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate–fumarate–succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium.

Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: Effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids

During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes.

Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.

Ubiquinone-binding Site Mutations in the Saccharomyces cerevisiae Succinate Dehydrogenase Generate Superoxide and Lead to the Accumulation of Succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo. We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: Compartmentation of astrocyte metabolism

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2- 13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the "pet" operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild-type strain (K-12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.