Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autoren

  • Christoph Wittmann
  • Jan Weber
  • Eriola Betiku
  • Jens Krömer
  • Daniela Böhm
  • Ursula Rinas

Externe Organisationen

  • Universität des Saarlandes
  • Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
Forschungs-netzwerk anzeigen

Details

OriginalspracheEnglisch
Seiten (von - bis)375-384
Seitenumfang10
FachzeitschriftJournal of biotechnology
Jahrgang132
Ausgabenummer4
PublikationsstatusVeröffentlicht - 1 Dez. 2007
Extern publiziertJa

Abstract

The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:pλFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.

ASJC Scopus Sachgebiete

Zitieren

Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli. / Wittmann, Christoph; Weber, Jan; Betiku, Eriola et al.
in: Journal of biotechnology, Jahrgang 132, Nr. 4, 01.12.2007, S. 375-384.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Wittmann C, Weber J, Betiku E, Krömer J, Böhm D, Rinas U. Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli. Journal of biotechnology. 2007 Dez 1;132(4):375-384. doi: 10.1016/j.jbiotec.2007.07.495
Wittmann, Christoph ; Weber, Jan ; Betiku, Eriola et al. / Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli. in: Journal of biotechnology. 2007 ; Jahrgang 132, Nr. 4. S. 375-384.
Download
@article{82b3b1d3fd9441b9b1b460fd96936eaa,
title = "Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli",
abstract = "The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:pλFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.",
keywords = "C metabolic flux, Adenylate energy charge, Cellular stress, Human basic fibroblast growth factor",
author = "Christoph Wittmann and Jan Weber and Eriola Betiku and Jens Kr{\"o}mer and Daniela B{\"o}hm and Ursula Rinas",
note = "Funding Information: Eriola Betiku gratefully acknowledges financial support by the Deutscher Akademischer Austauschdienst (DAAD). Jens Kr{\"o}mer acknowledges financial support of the BASF AG (Ludwigshafen, Germany).",
year = "2007",
month = dec,
day = "1",
doi = "10.1016/j.jbiotec.2007.07.495",
language = "English",
volume = "132",
pages = "375--384",
journal = "Journal of biotechnology",
issn = "0168-1656",
publisher = "Elsevier",
number = "4",

}

Download

TY - JOUR

T1 - Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli

AU - Wittmann, Christoph

AU - Weber, Jan

AU - Betiku, Eriola

AU - Krömer, Jens

AU - Böhm, Daniela

AU - Rinas, Ursula

N1 - Funding Information: Eriola Betiku gratefully acknowledges financial support by the Deutscher Akademischer Austauschdienst (DAAD). Jens Krömer acknowledges financial support of the BASF AG (Ludwigshafen, Germany).

PY - 2007/12/1

Y1 - 2007/12/1

N2 - The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:pλFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.

AB - The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:pλFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.

KW - C metabolic flux

KW - Adenylate energy charge

KW - Cellular stress

KW - Human basic fibroblast growth factor

UR - http://www.scopus.com/inward/record.url?scp=35748959651&partnerID=8YFLogxK

U2 - 10.1016/j.jbiotec.2007.07.495

DO - 10.1016/j.jbiotec.2007.07.495

M3 - Article

C2 - 17689798

AN - SCOPUS:35748959651

VL - 132

SP - 375

EP - 384

JO - Journal of biotechnology

JF - Journal of biotechnology

SN - 0168-1656

IS - 4

ER -