Exacerbation of Substrate Toxicity by IPTG in Escherichia coli
BL21(DE3) Carrying a Synthetic Metabolic Pathway

J 2015

Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway

DVOŘÁK, Pavel, Lukáš CHRÁST, Pablo Ivan NIKEL, Radek FEDR, Karel SOUČEK et. al.

Základní údaje

Originální název

Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway

Autoři

DVOŘÁK, Pavel (203 Česká republika, domácí), Lukáš CHRÁST (203 Česká republika, domácí), Pablo Ivan NIKEL (32 Argentina), Radek FEDR (203 Česká republika), Karel SOUČEK (203 Česká republika, domácí), Miroslava SEDLÁČKOVÁ (203 Česká republika, domácí), Radka CHALOUPKOVÁ (203 Česká republika, domácí), Víctor LORENZO DE (724 Španělsko), Zbyněk PROKOP (203 Česká republika, domácí) a Jiří DAMBORSKÝ (203 Česká republika, garant, domácí)

Vydání

Microbial Cell Factories, London, BioMed Central, 2015, 1475-2859

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 3.744

Kód RIV

RIV/00216224:14310/15:00081393

Organizační jednotka

Přírodovědecká fakulta

DOI

http://dx.doi.org/10.1186/s12934-015-0393-3

UT WoS

000367047300002

Klíčová slova anglicky

Metabolic burden; Substrate toxicity; Escherichia coli; Heterologous metabolic pathway; Isopropyl beta-D-1-thiogalactopyranoside; Lactose; 1.2.3-trichloropropane; Metabolic engineering

Štítky

AKR, EL OK, podil, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 21. 3. 2017 07:57, prof. Mgr. Jiří Damborský, Dr.

Anotace

V originále

Background: Heterologous expression systems based on promoters inducible with isopropyl-beta-D-1- thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacIQ/PlacUV5-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, sidereactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. Results: The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer’s chemical properties. Conclusions: IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect.

Návaznosti

GAP503/12/0572, projekt VaV

Název: Konstrukce syntetické metabolické dráhy pro degradaci důležitého environmentálního polutantu proteinovým a metabolickým inženýrstvím

Investor: Grantová agentura ČR, Konstrukce syntetické metabolické dráhy pro degradaci důležitého environmentlního polutantu proteinovým a metabolickým inženýrstvím

LM2011028, projekt VaV

Název: RECETOX ? Národní infrastruktura pro výzkum toxických látek v prostředí

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, RECETOX Národní infrastruktura pro výzkum toxických látek v prostředí

LO1214, projekt VaV

Název: Centrum pro výzkum toxických látek v prostředí (Akronym: RECETOX)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Centrum pro výzkum toxických látek v prostředí

Citovat

DVOŘÁK, Pavel, Lukáš CHRÁST, Pablo Ivan NIKEL, Radek FEDR, Karel SOUČEK, Miroslava SEDLÁČKOVÁ, Radka CHALOUPKOVÁ, Víctor LORENZO DE, Zbyněk PROKOP a Jiří DAMBORSKÝ. Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway. Microbial Cell Factories. London: BioMed Central, 2015, roč. 14, December, s. "nestrankovano", 15 s. ISSN 1475-2859. Dostupné z: https://dx.doi.org/10.1186/s12934-015-0393-3.

@article{1322825,
   author = {Dvořák, Pavel and Chrást, Lukáš and Nikel, Pablo Ivan and Fedr, Radek and Souček, Karel and Sedláčková, Miroslava and Chaloupková, Radka and Lorenzo de, Víctor and Prokop, Zbyněk and Damborský, Jiří},
   article_location = {London},
   article_number = {December},
   doi = {http://dx.doi.org/10.1186/s12934-015-0393-3},
   keywords = {Metabolic burden; Substrate toxicity; Escherichia coli; Heterologous metabolic pathway; Isopropyl beta-D-1-thiogalactopyranoside; Lactose; 1.2.3-trichloropropane; Metabolic engineering},
   language = {eng},
   issn = {1475-2859},
   journal = {Microbial Cell Factories},
   title = {Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway},
   url = {http://loschmidt.chemi.muni.cz/peg/wp-content/uploads/2015/12/Dvorak_2015MCF.pdf},
   volume = {14},
   year = {2015}
}

TY  - JOUR
ID  - 1322825
AU  - Dvořák, Pavel - Chrást, Lukáš - Nikel, Pablo Ivan - Fedr, Radek - Souček, Karel - Sedláčková, Miroslava - Chaloupková, Radka - Lorenzo de, Víctor - Prokop, Zbyněk - Damborský, Jiří
PY  - 2015
TI  - Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway
JF  - Microbial Cell Factories
VL  - 14
IS  - December
SP  - "nestrankovano"
EP  - "nestrankovano"
PB  - BioMed Central
SN  - 14752859
KW  - Metabolic burden
KW  - Substrate toxicity
KW  - Escherichia coli
KW  - Heterologous metabolic pathway
KW  - Isopropyl beta-D-1-thiogalactopyranoside
KW  - Lactose
KW  - 1.2.3-trichloropropane
KW  - Metabolic engineering
UR  - http://loschmidt.chemi.muni.cz/peg/wp-content/uploads/2015/12/Dvorak_2015MCF.pdf
L2  - http://loschmidt.chemi.muni.cz/peg/wp-content/uploads/2015/12/Dvorak_2015MCF.pdf
N2  - Background: Heterologous expression systems based on promoters inducible with isopropyl-beta-D-1- thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacIQ/PlacUV5-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, sidereactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. Results: The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer’s chemical properties. Conclusions: IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect.
ER  -

DVOŘÁK, Pavel, Lukáš CHRÁST, Pablo Ivan NIKEL, Radek FEDR, Karel SOUČEK, Miroslava SEDLÁČKOVÁ, Radka CHALOUPKOVÁ, Víctor LORENZO DE, Zbyněk PROKOP a Jiří DAMBORSKÝ. Exacerbation of Substrate Toxicity by IPTG in Escherichia coli BL21(DE3) Carrying a Synthetic Metabolic Pathway. \textit{Microbial Cell Factories}. London: BioMed Central, 2015, roč.~14, December, s.~''nestrankovano'', 15 s. ISSN~1475-2859. Dostupné z: https://dx.doi.org/10.1186/s12934-015-0393-3.

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