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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.

ABSTRACT: 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.

SUBMITTER: Zhao C

PROVIDER: S-EPMC5440719 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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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 Chunhua C Lin Zhao Z Dong Hongjun H Zhang Yanping Y Li Yin Y

Applied and environmental microbiology 20170517 11

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 <i>Escherichia coli</i> PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the <i>pykA</i> gene to increase the level of its substrate, PEP, and thereby increased the yield of end pro ...[more]

PMID: 28363967

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Project description:Gene expression profiles of Escherichia coli, grown anaerobically, with or without Acacia mearnsii (Black wattle) extract were compared to identify tannin-resistance strategies. The cell envelope stress protein, spy, and the multidrug transporter-encoding mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin-sensitive than their wild-type counterparts. Experiment Overall Design: E. coli BW13711 cells were grown anaerobically in continuous culture (d = 0.14h-1) in the presence and absence of Acacia mearnsii (Black Wattle) tannin extract (WTE) at 37Â°C. Under both steady state conditions, E. coli reached an OD600 of ~0.2. The pH at steady-state was 5.7 and 5.3 without and with WTE, respectively. These conditions indicated that the presence of tannins in the medium did not inhibit the growth of E. coli. E. coli BW13711 cells were harvested from continuous culture after steady-state conditions were reached. Duplicate samples were collected after an additional four volume turnover of the culture medium and total RNA was isolated from these samples using the RNeasy mini kit (Qiagen Inc., Valencia, CA). The presence of residual DNA was checked by PCR using the primers for amplification of cca. If necessary, the remaining DNA was removed by RQ1 RNase-Free DNase (Promega corp, Madison, WI) according to the manufacturerâs instructions. E. coli whole genome transcriptional profiling was performed by hybridization on the GeneChipÂ® E. coli Antisense Genome Array (Affymetrix Inc., Santa Clara, CA) according to the manufacturerâs instructions. Microarray data were analyzed using MicroArray Suite 5.0 (Affymetrix Inc.) and GeneSpring 5.0 (Silicon Genetics, Redwood City, CA) software. The 95% confidence interval of the WTE array duplicates demonstrated that genes >1.9 times up- or down-regulated could be considered as significantly differentially regulated. After removing significantly up- or down-regulated genes which had large variability in expression on the duplicate arrays, it was found that in total 1,498 genes were up- or down-regulated

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Dataset Information

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

Publications

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

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