Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

Meza, Eugenio; Becker, Jörg; Bolívar, Francisco; Gosset, Guillermo; Wittmann, Christoph

doi:10.1186/1475-2859-11-127

Cited by 71 publications

(59 citation statements)

References 56 publications

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“…This is consistent with the previous recognition that PykF is the main pyruvate kinase in E. coli (25,26). However, under anaerobic conditions, the activity of PykF accounted for 70% of the total pyruvate …”

Section: Resultssupporting

confidence: 93%

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Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Zhao

Dong

et al. 2017

Appl Environ Microbiol

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Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli. PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA::Tn5 mutant in a screen for increased yield of an end product derived from pyruvate (n-butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n-butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products.KEYWORDS ATP, anaerobic condition, Escherichia coli, physiological role, PykA I ndustrial fermentation aims to produce valuable products from cheap feedstocks by utilizing the diverse functions of microbes. Products such as antibiotics, amino acids, and vitamins are mostly produced through aerobic fermentations, while products such as alcohols (ethanol, butanol, butanediol) are mainly produced through anaerobic fermentation (1-3). Organic acids such as lactic acid and succinic acid are also produced through anaerobic fermentation because higher yields could be obtained (4,5).During anaerobic fermentation, the reducing power is directed mostly to producing synthesis rather than to oxidization, resulting in a higher yield of target products. In addition, the low energy level due to the absence of oxidative phosphorylation often leads to a higher glycolysis rate, resulting in higher productivity (6). However, as the available ATP in anaerobic fermentation can be generated only from substrate-level phosphorylation, the biomass concentration in anaerobic fermentation is usually much

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Section: Resultssupporting

confidence: 93%

“…In the aerobic glycolysis pathway of E. coli, PykF is believed to be the major contributor of pyruvate kinase activity, while PykA contributes little (25,26). Our enzymatic assay confirmed that PykA does contribute very little (7%) to total pyruvate kinase activity (Table 4).…”

Section: Discussionsupporting

confidence: 68%

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Zhao

Dong

et al. 2017

Appl Environ Microbiol

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“…A knock-out of the phosphotransferase system is known in the literature (Escalante et al, 2012) allowing a change of glucose uptake mechanism especially if combined with overexpression of hexose permease (Hernandez-Montalvo et al, 2003). Also, combinations of PTS and PYK knock-out (Lee et al, 2005) have been applied successfully, especially in respect to the production of AAAs (Gosset et al, 1996, Meza et al, 2012, Papagianni, 2012. Also very recently the knock-out of PTS and PYK by means of ptsH, ptsI, crr, and pykF has been described and applied for ccMA production from pHBA (Sengupta et al, 2015).…”

Section: Metabolic Peculiarities Of E Coli Hinder Knock-out Strategiesmentioning

confidence: 99%

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

Averesch¹

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Aromatic building blocks are amongst the most important bulk feedstocks in the chemical industry. As these compounds are commonly derived from petrochemistry, obtaining them is becoming more and more a matter of costs and sustainability. Biochemistry gives rise to a wealth of compounds that can potentially replace current petroleum-based chemicals or be used for novel materials. The aromatic compounds para-aminobenzoic acid (pABA) and para-hydroxybenzoic acid (pHBA) and the aromatics derived compound cis,cismuconic acid (ccMA) can be precursors for, but are not limited to, terephthalic or/and adipic acid. These are essential feedstocks for the production of PET and nylon. The three compounds can be derived from the shikimate pathway, an anabolic pathway leading to the biosynthesis of aromatic amino acids, present in certain prokaryotes and eukaryotes, including fungi. By combination of metabolic modelling with genetic engineering, a microbial production system based on the yeast Saccharomyces cerevisiae can be designed, which effectively channels flux into the target compounds.In order to develop a competitive bio-based process, yields, titers and rates need to be maximized. While productivity or rates in a process can be altered using genetic engineering, carbon yield, and pathway feasibility are stoichiometrically and thermodynamically predetermined. Both limitations need to be considered when designing a microbial production system. For formation of adipic acid and precursors many bio-based routes exists. More rational than just picking one for in vivo studies rather all available biochemical pathways were examined in silico using metabolic modelling. To compare theoretical yields and reaction thermodynamics an interface that allowed network-embedded thermodynamic analysis of elementary flux modes was developed. This allowed distinguishing between thermodynamically feasible and infeasible flux distributions. Feasible maximum theoretical product carbon yields were substantially different in E. coli and S. cerevisiae metabolic models and ranged from 32% to 92%. Further, many pathways appeared to be restricted by a thermodynamic equilibrium lying on the substrate side, some even infeasible.The only routes that deliver significant product yields and were thermodynamically favoured were shikimate pathway based. Being currently of strong scientific interest, recent implementations of these pathways in E. coli and S. cerevisiae were evaluated and strain construction strategies optimized, using the concept of constrained minimal cut-sets.Especially in S. cerevisiae a single non-obvious knock-out target allowed coupling of growth to product formation; in particular, the deletion of the pyruvate kinase reaction resulted in a minimum yield constraint of 28%.Though unique to shikimate pathway, this strategy is transferrable to other products which are derived from chorismate and also involve the formation of pyruvate as a by-product. This applies to pHBA. With further optimizations, the strategy was applied in...

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“…Furthermore, PTS inactivation revealed a carbon recycling response between PEP and OAA combined with a reduced glycolytic flux. When these strains were transformed with plasmids encoding enzymes to promote the production of L-PHE, a 19-, 14-, and 25-fold increase on the yield of this amino acid was observed for the PTS, PTS − pykA, and PTS − pykF mutants, respectively [33]. Targeted proteomics and metabolite profiling analyses are also very valuable to provide feedback about expression systems used in the production of AAA.…”

Section: (S)-reticuline ((1s)-1-[(3-hydroxy-4-methoxyphenyl)methyl]-6mentioning

confidence: 99%

“…Additionally, high PEP availability has been achieved by modulation of the carbon flux from PEP to the TCA caused by the inactivation of one or both of the PYR kinases [33,34], as well as improving the recycling of PYR to PEP by a plasmid-encoded copy of PEP synthetase [35][36][37]. The overexpression of pckA, in combination with an enhanced carbon flow through the glyoxylate shunt, has also been proposed as a strategy to increase the yield of aromatic compounds [38,39].…”

Section: Introductionmentioning

confidence: 99%

Engineering

et al. 2014

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The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.

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Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

Cited by 71 publications

References 56 publications

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

Engineering

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