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Title: Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC

Abstract

ABSTRACT Limonene, a prime component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its possibility as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain ofEscherichia coliand found a mutation inahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We demonstrate that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC L177Q) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We reveal that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-typeE. colicells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.

Authors:
 [1];  [2];  [3];  [1];  [1];  [4];  [1];  [4];  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Total New Energies USA, Inc., Emeryville, CA (United States)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1512098
DOE Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 81; Journal Issue: 14; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Chubukov, Victor, Mingardon, Florence, Schackwitz, Wendy, Baidoo, Edward E. K., Alonso-Gutierrez, Jorge, Hu, Qijun, Lee, Taek Soon, Keasling, Jay D., and Mukhopadhyay, Aindrila. Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC. United States: N. p., 2015. Web. doi:10.1128/AEM.01102-15.
Chubukov, Victor, Mingardon, Florence, Schackwitz, Wendy, Baidoo, Edward E. K., Alonso-Gutierrez, Jorge, Hu, Qijun, Lee, Taek Soon, Keasling, Jay D., & Mukhopadhyay, Aindrila. Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC. United States. doi:10.1128/AEM.01102-15.
Chubukov, Victor, Mingardon, Florence, Schackwitz, Wendy, Baidoo, Edward E. K., Alonso-Gutierrez, Jorge, Hu, Qijun, Lee, Taek Soon, Keasling, Jay D., and Mukhopadhyay, Aindrila. Fri . "Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC". United States. doi:10.1128/AEM.01102-15. https://www.osti.gov/servlets/purl/1512098.
@article{osti_1512098,
title = {Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC},
author = {Chubukov, Victor and Mingardon, Florence and Schackwitz, Wendy and Baidoo, Edward E. K. and Alonso-Gutierrez, Jorge and Hu, Qijun and Lee, Taek Soon and Keasling, Jay D. and Mukhopadhyay, Aindrila},
abstractNote = {ABSTRACT Limonene, a prime component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its possibility as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain ofEscherichia coliand found a mutation inahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We demonstrate that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpCL177Q) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We reveal that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-typeE. colicells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.},
doi = {10.1128/AEM.01102-15},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 14,
volume = 81,
place = {United States},
year = {2015},
month = {5}
}

Works referenced in this record:

Engineering of Bacterial Methyl Ketone Synthesis for Biofuels
journal, October 2011

  • Goh, Ee-Been; Baidoo, Edward E. K.; Keasling, Jay D.
  • Applied and Environmental Microbiology, Vol. 78, Issue 1
  • DOI: 10.1128/AEM.06785-11

Efficient conversion of pure and mixed terpene feedstocks to high density fuels
journal, July 2012


Engineering microbial biofuel tolerance and export using efflux pumps
journal, January 2011

  • Dunlop, Mary J.; Dossani, Zain Y.; Szmidt, Heather L.
  • Molecular Systems Biology, Vol. 7, Issue 1
  • DOI: 10.1038/msb.2011.21

ORANGE CRUSH: Tight -LIMONENE SUPPLIES put the squeeze on makers of citrus-based cleaners
journal, April 2011


Limonene: a versatile chemical of the bioeconomy
journal, January 2014

  • Ciriminna, Rosaria; Lomeli-Rodriguez, Monica; Demma Carà, Piera
  • Chem. Commun., Vol. 50, Issue 97
  • DOI: 10.1039/C4CC06147K

Hydroperoxides in oxidized d-limonene identified as potent contact allergens
journal, February 1994

  • Karlberg, A. -T.; Shao, L. P.; Nilsson, U.
  • Archives of Dermatological Research, Vol. 286, Issue 2
  • DOI: 10.1007/BF00370734

Limonene hydroperoxide analogues differ in allergenic activity
journal, December 2008


Selection and optimization of microbial hosts for biofuels production
journal, November 2008

  • Fischer, Curt R.; Klein-Marcuschamer, Daniel; Stephanopoulos, Gregory
  • Metabolic Engineering, Vol. 10, Issue 6, p. 295-304
  • DOI: 10.1016/j.ymben.2008.06.009

Inhibition of foodborne pathogen bacteria by essential oils extracted from citrus fruits cultivated in Sicily
journal, August 2012


Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae
journal, May 2012

  • Brennan, Timothy C. R.; Turner, Christopher D.; Krömer, Jens O.
  • Biotechnology and Bioengineering, Vol. 109, Issue 10
  • DOI: 10.1002/bit.24536

Changes in Membrane Fatty Acids Composition of Microbial Cells Induced by Addiction of Thymol, Carvacrol, Limonene, Cinnamaldehyde, and Eugenol in the Growing Media
journal, April 2006

  • Di Pasqua, Rosangela; Hoskins, Nikki; Betts, Gail
  • Journal of Agricultural and Food Chemistry, Vol. 54, Issue 7
  • DOI: 10.1021/jf052722l

Fast and accurate short read alignment with Burrows-Wheeler transform
journal, May 2009


Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases
journal, January 2005


Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection
journal, February 2006

  • Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki
  • Molecular Systems Biology, Vol. 2, Article No. 2006.0008
  • DOI: 10.1038/msb4100050

j5 DNA Assembly Design Automation Software
journal, December 2011

  • Hillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D.
  • ACS Synthetic Biology, Vol. 1, Issue 1, p. 14-21
  • DOI: 10.1021/sb2000116

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production
journal, September 2013


BglBrick vectors and datasheets: A synthetic biology platform for gene expression
journal, January 2011

  • Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong
  • Journal of Biological Engineering, Vol. 5, Issue 1
  • DOI: 10.1186/1754-1611-5-12

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli
journal, October 2014

  • Dip, Phat Vinh; Kamariah, Neelagandan; Subramanian Manimekalai, Malathy Sony
  • Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 11
  • DOI: 10.1107/S1399004714019233

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d -Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane
journal, March 2013

  • Brennan, Timothy C. R.; Krömer, Jens O.; Nielsen, Lars K.
  • Applied and Environmental Microbiology, Vol. 79, Issue 12
  • DOI: 10.1128/AEM.00463-13

Analysis of the Link between Enzymatic Activity and Oligomeric State in AhpC, a Bacterial Peroxiredoxin ,
journal, August 2005

  • Parsonage, Derek; Youngblood, Derek S.; Sarma, Ganapathy N.
  • Biochemistry, Vol. 44, Issue 31
  • DOI: 10.1021/bi050448i

Carbon- and Oxygen-Centered Radicals Are Equally Important Haptens of Allylic Hydroperoxides in Allergic Contact Dermatitis
journal, August 2008

  • Johansson, Staffan; Giménez-Arnau, Elena; Grøtli, Morten
  • Chemical Research in Toxicology, Vol. 21, Issue 8
  • DOI: 10.1021/tx800104c

Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases
journal, September 1983


Oxidative stress
journal, April 1999


Exogenous ergosterol protects Saccharomyces cerevisiae from d -limonene stress
journal, November 2012

  • Liu, J.; Zhu, Y.; Du, G.
  • Journal of Applied Microbiology, Vol. 114, Issue 2
  • DOI: 10.1111/jam.12046

Monoterpene biosynthesis pathway construction in Escherichia coli
journal, September 2003


Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO 2 and H 2 O
journal, January 2014

  • Halfmann, Charles; Gu, Liping; Zhou, Ruanbao
  • Green Chem., Vol. 16, Issue 6
  • DOI: 10.1039/C3GC42591F

The compatibility of potential bioderived fuels with Jet A-1 aviation kerosene
journal, April 2014


Determination of allergenic hydroperoxides in essential oils using gas chromatography with electron ionization mass spectrometry: Gas Chromatography
journal, March 2014

  • Rudbäck, Johanna; Ramzy, Ahmed; Karlberg, Ann-Therese
  • Journal of Separation Science, Vol. 37, Issue 8
  • DOI: 10.1002/jssc.201300843

Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012

  • Ham, T. S.; Dmytriv, Z.; Plahar, H.
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks531

Zur chemischen und sterischen Selektivität der photosensibilisierten O2-Übertragung auf (+)-Limonen und (+)-Carvomenthen
journal, July 1964

  • Schenck, Günther O.; Gollnick, Klaus; Buchwald, Gustav
  • Justus Liebigs Annalen der Chemie, Vol. 674, Issue 1
  • DOI: 10.1002/jlac.19646740111

The Sequence Alignment/Map format and SAMtools
journal, June 2009


Characterization of the hydroperoxides derived from singlet oxygen oxidation of ( + )-limonene
journal, January 1981


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.
journal, August 1995

  • Ferrante, A. A.; Augliera, J.; Lewis, K.
  • Proceedings of the National Academy of Sciences, Vol. 92, Issue 17
  • DOI: 10.1073/pnas.92.17.7617

Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration
journal, April 2014


2,2-Diphenyl-1-picrylhydrazyl as a screening tool for recombinant monoterpene biosynthesis
journal, January 2013

  • Behrendorff, James BYH; Vickers, Claudia E.; Chrysanthopoulos, Panagiotis
  • Microbial Cell Factories, Vol. 12, Issue 1
  • DOI: 10.1186/1475-2859-12-76

Mechanisms of Solvent Tolerance in Gram-Negative Bacteria
journal, October 2002