Engineering of phosphoenolpyruvate: carbohydrate phosphotransferase system increased acetate assimilation in Escherichia coli.
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ABSTRACT:
SUBMITTER: Gu P
PROVIDER: S-EPMC6372417 | biostudies-literature | 2019 Mar
REPOSITORIES: biostudies-literature
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Project description:BackgroundShikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.ResultsBatch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (mu) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the mu decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in mu, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).ConclusionsThe effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.
| S-EPMC2873404 | biostudies-literature
Project description:In Escherichia coli, the transport and phosphorylation of glucose is mainly accomplished by the phosphoenolpyruvate-dependent glucose-specific phosphotransferase system (PTSGlc), which is, therefore, frequently selected as a target for engineering to increase the intracellular level of phosphoenolpyruvate. Here we characterized the effects of a low glucose concentration on the growth, glucose consumption, and acetate secretion of individual strains with a single PTSGlc mutation. We found that most mutants accumulated similar amounts of biomass, consumed glucose at lower rates, and secreted less acetate compared with the wild-type parental strain. The exception was the growth-impaired strain MG1655I harboring a ptsI deletion. In summary, the fermentation performance of mutant strains under 5 g/L glucose was obviously different with those strains under 20 g/L glucose. This study is a good complement to the knowledge of PTSGlc in E. coli and indicates that engineering the components of PTSGlc should be carefully optimized, particularly during fermentation in the presence of low concentrations of glucose.
| S-EPMC6346689 | biostudies-literature
Project description:BackgroundEfficient production of SA in Escherichia coli has been achieved by modifying key genes of the central carbon metabolism and SA pathway, resulting in overproducing strains grown in batch- or fed-batch-fermentor cultures using a complex broth including glucose and YE. In this study, we performed a GTA to identify those genes significantly upregulated in an engineered E. coli strain, PB12.SA22, in mid EXP (5 h), early STA (STA1, 9 h), and late STA (STA2, 44 h) phases, grown in complex fermentation broth in batch-fermentor cultures.ResultsGrowth of E. coli PB12.SA22 in complex fermentation broth for SA production resulted in an EXP growth during the first 9 h of cultivation depending of supernatant available aromatic amino acids provided by YE because, when tryptophan was totally consumed, cells entered into a second, low-growth phase (even in the presence of glucose) until 26 h of cultivation. At this point, glucose was completely consumed but SA production continued until the end of the fermentation (50 h) achieving the highest accumulation (7.63 g/L of SA). GTA between EXP/STA1, EXP/STA2 and STA1/STA2 comparisons showed no significant differences in the regulation of genes encoding enzymes of central carbon metabolism as in SA pathway, but those genes encoding enzymes involved in sugar, amino acid, nucleotide/nucleoside, iron and sulfur transport; amino acid catabolism and biosynthesis; nucleotide/nucleoside salvage; acid stress response and modification of IM and OM were upregulated between comparisons.ConclusionsGTA during SA production in batch-fermentor cultures of strain PB12.SA22 grown in complex fermentation broth during the EXP, STA1 and STA2 phases was studied. Significantly, upregulated genes during the EXP and STA1 phases were associated with transport, amino acid catabolism, biosynthesis, and nucleotide/nucleoside salvage. In STA2, upregulation of genes encoding transporters and enzymes involved in the synthesis and catabolism of Arg suggests that this amino acid could have a key role in the fuelling of carbon toward SA synthesis, whereas upregulation of genes involved in pH stress response, such as membrane modifications, suggests a possible response to environmental conditions imposed on the cell at the end of the fermentation.
| S-EPMC4015609 | biostudies-literature
Project description:Chemotaxis allows bacteria to follow gradients of nutrients, environmental stimuli, and signaling molecules, optimizing bacterial growth and survival. Escherichia coli has long served as a model of bacterial chemotaxis, and the signal processing by the core of its chemotaxis pathway is well understood. However, most of the research so far has focused on one branch of chemotactic signaling, in which ligands bind to periplasmic sensory domains of transmembrane chemoreceptors and induce a conformational change that is transduced across the membrane to regulate activity of the receptor-associated kinase CheA. Here we quantitatively characterize another, receptor-independent branch of chemotactic signaling that is linked to the sugar uptake through a large family of phosphotransferase systems (PTSs). Using in vivo characterization of intracellular signaling and protein interactions, we demonstrate that signals from cytoplasmic PTS components are transmitted directly to the sensory complexes formed by chemoreceptors, CheA and an adapter protein CheW. We further conclude that despite different modes of sensing, the PTS- and receptor-mediated signals have similar regulatory effects on the conformation of the sensory complexes. As a consequence, both types of signals become integrated and undergo common downstream processing including methylation-dependent adaptation. We propose that such mode of signaling is essential for efficient chemotaxis to PTS substrates and may be common to most bacteria.
| S-EPMC3409764 | biostudies-literature
Project description:Csr is a conserved global regulatory system, which uses the sequence-specific RNA-binding protein CsrA to activate or repress gene expression by binding to mRNA and altering translation, stability and/or transcript elongation. In Escherichia coli, CsrA activity is regulated by two sRNAs, CsrB and CsrC, which bind to multiple CsrA dimers, thereby sequestering this protein away from its mRNA targets. Turnover of CsrB/C sRNAs is tightly regulated by a GGDEF-EAL domain protein, CsrD, which targets them for cleavage by RNase E. Here, we show that EIIA(Glc) of the glucose-specific PTS system is also required for the normal decay of these sRNAs and that it acts by binding to the EAL domain of CsrD. Only the unphosphorylated form of EIIA(Glc) bound to CsrD?in vitro and was capable of activating CsrB/C turnover in vivo. Genetic studies confirmed that this mechanism couples CsrB/C sRNA decay to the availability of a preferred carbon source. These findings reveal a new physiological influence on the workings of the Csr system, a novel function for the EAL domain, and an important new way in which EIIA(Glc) shapes global regulatory circuitry in response to nutritional status.
| S-EPMC4888959 | biostudies-literature
Project description:AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr- EI- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.
| S-EPMC8462991 | biostudies-literature
Project description:The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.
| S-EPMC4054256 | biostudies-literature
Project description:Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACB and H16 ΔnagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.
| S-EPMC3127587 | biostudies-literature
Project description:1. A method is described for measuring the rate of phosphoenolpyruvate-dependent phosphotransferase activity for a variety of hexoses in toluene-treated suspensions of Escherichia coli. 2. The specific activities of the phosphotransferases that catalyse the phosphorylation of hexoses are greatly affected by the carbon source for growth. 3. In all strains of E. coli tested, fructose phosphotransferase activity is induced by growth on fructose. 4. Strains of E. coli differ greatly in the rate at which they phosphorylate glucose, but all strains possess at least a low glucose phosphotransferase activity under any tested condition of growth. Glucose phosphotransferase activity is further induced by growth on glucose; this does not occur in a mutant that lacks the ability to take up methyl alpha-d-[(14)C]glucopyranoside and hence grows poorly on glucose. 5. When growing on fructose, two strains of E. coli synthesize the inducible glucose phosphotransferase system gratuitously, and to specific activities higher than observed during growth on glucose. A phosphotransferase catalysing the phosphorylation of mannose is similarly induced.
| S-EPMC1174022 | biostudies-other
Project description:In Vibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR::lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc Furthermore, the regulation of tfoR and chb expression by EIIAglc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCE The EIIAglc protein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways in V. cholerae with EIIAglc The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglc inhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways in V. cholerae and suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.
| S-EPMC5573088 | biostudies-literature