Development of a native Escherichia coli induction system for ionic liquid tolerance

Marijke Frederix; Kimmo Hütter; Jessica Leu; Tanveer S Batth; William J Turner; Thomas L Rüegg; Harvey W Blanch; Blake A Simmons; Paul D Adams; Jay D Keasling; Michael P Thelen; Mary J Dunlop; Christopher J Petzold; Aindrila Mukhopadhyay

doi:10.1371/journal.pone.0101115

Development of a native Escherichia coli induction system for ionic liquid tolerance

Marijke Frederix, Kimmo Hütter, Jessica Leu, Tanveer S Batth, William J Turner, Thomas L Rüegg, Harvey W Blanch, Blake A Simmons, Paul D Adams, Jay D Keasling, Michael P Thelen, Mary J Dunlop, Christopher J Petzold, Aindrila Mukhopadhyay

26 Citationer (Scopus)

Abstract

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

Originalsprog	Engelsk
Tidsskrift	PLOS ONE
Vol/bind	9
Udgave nummer	7
Sider (fra-til)	e101115
ISSN	1932-6203
DOI	https://doi.org/10.1371/journal.pone.0101115
Status	Udgivet - 1 jul. 2014

Adgang til dokumentet

10.1371/journal.pone.0101115

journal.pone.0101115&type=printableForlagets udgivne version, 1,4 MBLicens: CC BY

Citationsformater

Frederix, M., Hütter, K., Leu, J., Batth, T. S., Turner, W. J., Rüegg, T. L., Blanch, H. W., Simmons, B. A., Adams, P. D., Keasling, J. D., Thelen, M. P., Dunlop, M. J., Petzold, C. J., & Mukhopadhyay, A. (2014). Development of a native Escherichia coli induction system for ionic liquid tolerance. PLOS ONE, 9(7), e101115. https://doi.org/10.1371/journal.pone.0101115

@article{201bed4738ee4bf6a9bcd880043ad358,

title = "Development of a native Escherichia coli induction system for ionic liquid tolerance",

abstract = "The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains. ",

keywords = "Escherichia coli/drug effects, Escherichia coli Proteins/genetics, Ionic Liquids/pharmacology, Promoter Regions, Genetic, RNA, Bacterial/genetics, Transcriptome",

author = "Marijke Frederix and Kimmo H{\"u}tter and Jessica Leu and Batth, {Tanveer S} and Turner, {William J} and R{\"u}egg, {Thomas L} and Blanch, {Harvey W} and Simmons, {Blake A} and Adams, {Paul D} and Keasling, {Jay D} and Thelen, {Michael P} and Dunlop, {Mary J} and Petzold, {Christopher J} and Aindrila Mukhopadhyay",

year = "2014",

month = jul,

day = "1",

doi = "10.1371/journal.pone.0101115",

language = "English",

volume = "9",

pages = "e101115",

journal = "PLoS Computational Biology",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "7",

}

TY - JOUR

T1 - Development of a native Escherichia coli induction system for ionic liquid tolerance

AU - Frederix, Marijke

AU - Hütter, Kimmo

AU - Leu, Jessica

AU - Batth, Tanveer S

AU - Turner, William J

AU - Rüegg, Thomas L

AU - Blanch, Harvey W

AU - Simmons, Blake A

AU - Adams, Paul D

AU - Keasling, Jay D

AU - Thelen, Michael P

AU - Dunlop, Mary J

AU - Petzold, Christopher J

AU - Mukhopadhyay, Aindrila

PY - 2014/7/1

Y1 - 2014/7/1

N2 - The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

AB - The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

KW - Escherichia coli/drug effects

KW - Escherichia coli Proteins/genetics

KW - Ionic Liquids/pharmacology

KW - Promoter Regions, Genetic

KW - RNA, Bacterial/genetics

KW - Transcriptome

U2 - 10.1371/journal.pone.0101115

DO - 10.1371/journal.pone.0101115

M3 - Journal article

C2 - 24983352

SN - 1932-6203

VL - 9

SP - e101115

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 7

ER -

Development of a native Escherichia coli induction system for ionic liquid tolerance

Abstract

Adgang til dokumentet

Fingeraftryk

Citationsformater