title: Physiological and transcriptional responses of Saccharomyces cerevisiae to d-limonene show changes to the cell wall but not to the plasma membrane.
abstract: Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Timothy C R
authors.initials: TC
authors.lastname: Brennan
authors.forename: Jens O
authors.initials: JO
authors.lastname: Krömer
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
mesh_terms: D001557:Benzenesulfonates; D056804:Biofuels; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D002473:Cell Wall / Q000187:drug effects*; D053138:Cyclohexenes / Q000494:pharmacology*; D005227:Fatty Acids / Q000032:analysis; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D008401:Gas Chromatography-Mass Spectrometry; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D000077222:Limonene; D046228:Microarray Analysis; D018613:Microscopy, Confocal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D013729:Terpenes / Q000494:pharmacology*
chemical_list: D001557:Benzenesulfonates; D056804:Biofuels; D053138:Cyclohexenes; D005227:Fatty Acids; D013729:Terpenes; C007061:C.I. Fluorescent Brightening Agent 28; D000077222:Limonene
references.citation: Nat Protoc. 2006;1(5):2253-6
references.pmid: 17406464
references.citation: Free Radic Res. 1999 Oct;31(4):273-300
references.pmid: 10517533
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11206-10
references.pmid: 16837571
references.citation: Yeast. 2006 Feb;23(3):185-202
references.pmid: 16498706
references.citation: Appl Microbiol Biotechnol. 2009 Nov;85(2):253-63
references.pmid: 19756577
references.citation: Appl Environ Microbiol. 2003 Mar;69(3):1499-503
references.pmid: 12620835
references.citation: J Bacteriol. 1985 Mar;161(3):1195-1200
references.pmid: 3156123
references.citation: Science. 1980 Nov 21;210(4472):903-6
references.pmid: 7434003
references.citation: J Chem Ecol. 1990 Apr;16(4):1399-408
references.pmid: 24263736
references.citation: J Bacteriol. 1998 Aug;180(15):3735-40
references.pmid: 9683465
references.citation: Nat Protoc. 2006;1(6):2995-3000
references.pmid: 17406560
references.citation: Biochim Biophys Acta. 1999 Dec 6;1461(2):217-36
references.pmid: 10581358
references.citation: J Agric Food Chem. 2001 May;49(5):2552-62
references.pmid: 11368635
references.citation: J Bacteriol. 1998 Feb;180(4):938-44
references.pmid: 9473050
references.citation: Methods Enzymol. 1991;194:732-5
references.pmid: 2005820
references.citation: FEMS Microbiol Rev. 2000 Oct;24(4):469-86
references.pmid: 10978547
references.citation: Biochim Biophys Acta. 2000 Nov 23;1508(1-2):210-34
references.pmid: 11090827
references.citation: Fungal Genet Biol. 2005 Feb;42(2):165-77
references.pmid: 15670714
references.citation: Nat Genet. 2006 May;38(5):500-1
references.pmid: 16642009
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W193-200
references.pmid: 17478515
references.citation: Int J Pharm. 2007 Dec 10;345(1-2):9-25
references.pmid: 17945446
references.citation: J Appl Microbiol. 2013 Feb;114(2):482-91
references.pmid: 23082823
references.citation: Yeast. 2004 Apr 15;21(5):413-27
references.pmid: 15116342
references.citation: J Phys Chem B. 2012 Mar 29;116(12):3723-34
references.pmid: 22380931
references.citation: J Biol Chem. 1994 Mar 18;269(11):8022-8
references.pmid: 8132524
references.citation: Enzyme Microb Technol. 1990 Apr;12(4):281-91
references.pmid: 1366443
references.citation: Biotechnol Bioeng. 2012 Oct;109(10):2513-22
references.pmid: 22539043
references.citation: J Biol Chem. 2004 Apr 9;279(15):15183-95
references.pmid: 14739279
references.citation: Microb Cell Fact. 2011 Jan 28;10:4
references.pmid: 21276210
references.citation: J Biol Chem. 2001 Jun 8;276(23):19679-82
references.pmid: 11309404
references.citation: Biotechniques. 2003 Feb;34(2):374-8
references.pmid: 12613259
references.citation: Metab Eng. 2010 Jul;12(4):307-31
references.pmid: 20346409
references.citation: Genetics. 2011 Dec;189(4):1145-75
references.pmid: 22174182
references.citation: FEMS Microbiol Lett. 1998 Dec 1;169(1):191-7
references.pmid: 9851052
references.citation: FEMS Microbiol Lett. 2005 Feb 1;243(1):107-15
references.pmid: 15668008
references.citation: Mol Genet Genomics. 2001 Aug;265(6):1112-9
references.pmid: 11523784
references.citation: Appl Environ Microbiol. 1980 Aug;40(2):301-4
references.pmid: 16345609
references.citation: Biochim Biophys Acta. 2008 Jul;1783(7):1354-68
references.pmid: 18298957
references.citation: Appl Environ Microbiol. 2011 Sep;77(18):6400-8
references.pmid: 21784917
references.citation: J Gen Microbiol. 1983 May;129(5):1577-82
references.pmid: 6352860
references.citation: Mol Syst Biol. 2011 May 10;7:487
references.pmid: 21556065
references.citation: Microbiol Rev. 1995 Jun;59(2):201-22
references.pmid: 7603409
references.citation: Microbiol Mol Biol Rev. 2005 Jun;69(2):262-91
references.pmid: 15944456
references.citation: Arch Biochem Biophys. 2004 Jun 15;426(2):201-7
references.pmid: 15158670
references.citation: Biotechnol Bioeng. 2011 Aug;108(8):1883-92
references.pmid: 21391209
references.citation: J Bacteriol. 1998 Jan;180(1):163-6
references.pmid: 9422607
references.citation: Nature. 2005 Sep 29;437(7059):640-7
references.pmid: 16193038
references.citation: Biotechnol Lett. 2012 Aug;34(8):1505-9
references.pmid: 22526424
references.citation: J Antimicrob Chemother. 2004 Jul;54(1):46-55
references.pmid: 15201226
references.citation: Stat Appl Genet Mol Biol. 2004;3:Article3
references.pmid: 16646809
references.citation: J Biol Chem. 1998 Jan 2;273(1):85-91
references.pmid: 9417051
references.citation: J Agric Food Chem. 2007 Jul 25;55(15):6300-8
references.pmid: 17602646