Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of se...

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Autores principales:	Kyle A. Zingaro, Eleftherios Terry Papoutsakis
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a8e0919a32f741e09e11ead0b40cb17d
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spelling	oai:doaj.org-article:a8e0919a32f741e09e11ead0b40cb17d2021-11-15T15:39:12ZToward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance10.1128/mBio.00308-122150-7511https://doaj.org/article/a8e0919a32f741e09e11ead0b40cb17d2012-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00308-12https://doaj.org/toc/2150-7511ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.Kyle A. ZingaroEleftherios Terry PapoutsakisAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 5 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Kyle A. Zingaro Eleftherios Terry Papoutsakis Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
description	ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.
format	article
author	Kyle A. Zingaro Eleftherios Terry Papoutsakis
author_facet	Kyle A. Zingaro Eleftherios Terry Papoutsakis
author_sort	Kyle A. Zingaro
title	Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
title_short	Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
title_full	Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
title_fullStr	Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
title_full_unstemmed	Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance
title_sort	toward a semisynthetic stress response system to engineer microbial solvent tolerance
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/a8e0919a32f741e09e11ead0b40cb17d
work_keys_str_mv	AT kyleazingaro towardasemisyntheticstressresponsesystemtoengineermicrobialsolventtolerance AT eleftheriosterrypapoutsakis towardasemisyntheticstressresponsesystemtoengineermicrobialsolventtolerance
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Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

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