An insight into the role of phosphotransacetylase pta and the acetate-acetyl-CoA node in Escherichia coliReport as inadecuate




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Microbial Cell Factories

, 8:54

First Online: 24 October 2009Received: 31 July 2009Accepted: 24 October 2009

Abstract

BackgroundAcetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii the inhibition of cellular growth and protein production by acetate and iii the need for cofactor recycling namely redox coenzymes and free CoASH to sustain balanced growth and cellular homeostasis.

ResultsThis work analyzes the effect of mutations in the acetate excretion-assimilation pathways, acetyl-CoA synthethase acs and phosphotransacetylase pta, in E. coli BW25113 grown on glucose or acetate minimal media. Biomass and metabolite production, redox NADH-NAD and energy ATP state, enzyme activities and gene expression profiles related to the central metabolism were analyzed. The knock-out of pta led to a more altered phenotype than that of acs. Deletion of pta reduced the ability to grow on acetate as carbon source and strongly affected the expression of several genes related to central metabolic pathways.

ConclusionResults showed that pta limits biomass yield in aerobic glucose cultures, due to acetate production overflow metabolism and its inefficient use during glucose starvation. Deletion of pta severely impaired growth on acetate minimal medium and under anaerobiosis due to decreased acetyl-coenzyme A synthethase, glyoxylate shunt and gluconeogenic activities, leading to lower growth rate. When acetate is used as carbon source, the joint expression of pta and acs is crucial for growth and substrate assimilation, while pta deletion severely impaired anaerobic growth. Finally, at an adaptive level, pta deficiency makes the strain more sensitive to environmental changes and de-regulates the central metabolism.

List of abbreviations usedaceBAKglyoxylate shunt operon

aceEsubunit E1p of pyruvate dehydrogenase complex

aceFlipoate acetyltransferase subunit of pyruvate dehydrogenase complex

Ack ackacetate kinase

Acs acsacetyl-CoA synthetase

ADH adhEalcohol dehydrogenase

cAMPcyclic AMP

CoASHcoenzyme A

Cra-FruR cracatabolite repressor activator

Crp crpcatabolic repressor via cAMP receptor protein

Icdh icdisocitrate dehydrogenase

IcIR iclRglyoxylate shunt repressor

Icl aceAisocitrate lyase

iD-Ldh dldD-lactate dehydrogenase NAD-independent

IHF α subunit ihfAsubunit of integration host factor

iL-Ldh lldDL-lactate dehydrogenase NAD-independent

Ldh ldhAlactate dehydrogenase NAD-dependent

Lpd lpdlipoamide dehydrogenase, pyruvate dehydrogenase E3 monomer

MaeB maeBmalic enzyme NADP-requiring

Mdh mdhmalate dehydrogenase

MS aceBmalate synthase

OAAoxaloacetate

Pcc ppcPEP carboxylase

Pck pckPEP carboxykinase

Pdhpyruvate dehydrogenase complex

PdhR pdhRpyruvate dehydrogenase complex regulator

PEPphosphoenolpyruvate

Pfl pflBpyruvate formate lyase

PflA pflApyruvate formate lyase activating enzyme

polADNA polymerase I

PoxB poxBpyruvate oxidase

Pps ppsphosphoenolpyruvate synthase

Pta ptaphosphotransacetylase

PtsG ptsGsubunit of Enzyme II

Pyk pykFpykA: pyruvate kinase

RpoD or σsigma subunit of RNA polymerase

RpoS or σsigma subunit of RNA polymerase

Sdh sdhCsuccinate dehydrogenase membrane protein

SfcA sfcA-maeAmalic enzyme NAD-requiring

sucAsubunit of E1 0 component of 2-oxoglutarato dehydrogenase

TCAtricarboxylic acids cycle

Zwf zwfglucose 6-phosphate dehydrogenase.

Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-8-54 contains supplementary material, which is available to authorized users.

Sara Castaño-Cerezo, José M Pastor contributed equally to this work.

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Author: Sara Castaño-Cerezo - José M Pastor - Sergio Renilla - Vicente Bernal - José L Iborra - Manuel Cánovas

Source: https://link.springer.com/







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