Tolerance engineering in bacteria for the production of advanced biofuels and chemicals
2015; Elsevier BV; Volume: 23; Issue: 8 Linguagem: Inglês
10.1016/j.tim.2015.04.008
ISSN1878-4380
Autores Tópico(s)Biofuel production and bioconversion
Resumo•Microbial strains can be engineered to produce advanced biofuels and bulk chemicals that are inherently solvent-like in nature.•Solvents represent toxic end products for many host microbes.•Tolerance engineering focuses on developing microbes with improved robustness toward toxic or inhibitory end products.•Strains with improved tolerance can lead to improvement in productivity.•Cellular transporters that can export the toxic product provide an elegant mechanism to achieve greater tolerance and may also increase production. During microbial production of solvent-like compounds, such as advanced biofuels and bulk chemicals, accumulation of the final product can negatively impact the cultivation of the host microbe and limit the production levels. Consequently, improving solvent tolerance is becoming an essential aspect of engineering microbial production strains. Mechanisms ranging from chaperones to transcriptional factors have been used to obtain solvent-tolerant strains. However, alleviating growth inhibition does not invariably result in increased production. Transporters specifically have emerged as a powerful category of proteins that bestow tolerance and often improve production but are difficult targets for cellular expression. Here we review strain engineering, primarily as it pertains to bacterial solvent tolerance, and the benefits and challenges associated with the expression of membrane-localized transporters in improving solvent tolerance and production. During microbial production of solvent-like compounds, such as advanced biofuels and bulk chemicals, accumulation of the final product can negatively impact the cultivation of the host microbe and limit the production levels. Consequently, improving solvent tolerance is becoming an essential aspect of engineering microbial production strains. Mechanisms ranging from chaperones to transcriptional factors have been used to obtain solvent-tolerant strains. However, alleviating growth inhibition does not invariably result in increased production. Transporters specifically have emerged as a powerful category of proteins that bestow tolerance and often improve production but are difficult targets for cellular expression. Here we review strain engineering, primarily as it pertains to bacterial solvent tolerance, and the benefits and challenges associated with the expression of membrane-localized transporters in improving solvent tolerance and production. Metabolic engineering has been applied to various microbial hosts for the production of compounds spanning pharmaceuticals to bulk commodities. In every case, the discovery and optimization of the bioconversion pathway is an essential starting point. However, metabolic engineering for the sustained and reliable production of valuable metabolites is a complex and multifaceted effort [1Alper H. Stephanopoulos G. Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?.Nat. Rev. Microbiol. 2009; 7: 715-723Crossref PubMed Scopus (184) Google Scholar, 2Ling H. et al.Microbial tolerance engineering toward biochemical production: from lignocellulose to products.Curr. Opin. Biotechnol. 2014; 29: 99-106Crossref PubMed Scopus (3) Google Scholar, 3Nicolaou S.A. et al.A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation.Metab. Eng. 2010; 12: 307-331Crossref PubMed Scopus (214) Google Scholar, 4Peabody V. et al.Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.Curr. Opin. Chem. Eng. 2014; 6: 9-17Crossref Google Scholar, 5Mukhopadhyay A. et al.Control of stress tolerance in bacterial host organisms for bioproduction of fuels.in: Liu Z.L. Microbial Stress Tolerance for Biofuels. Springer, 2012: 209-238Crossref Google Scholar]. Host strain engineering that focuses on alternative aspects beyond the primary bioconversion pathway can lead to additional improvements in production levels and strain stability. These improvements are most relevant where productivity needs to be maximized to achieve economic value, as is the case for biofuels and bulk chemicals [6Yim H. et al.Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.Nat. Chem. Biol. 2011; 7: 445-452Crossref PubMed Scopus (289) Google Scholar, 7Erickson B. et al.Perspective on opportunities in industrial biotechnology in renewable chemicals.Biotechnol. J. 2012; 7: 176-185Crossref PubMed Scopus (61) Google Scholar, 8Ezeji T. et al.Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms.Appl. Microbiol. Biotechnol. 2010; 85: 1697-1712Crossref PubMed Scopus (125) Google Scholar]. One common aspect of strain engineering arises from the need to alleviate any cellular burden that limits production. In the case of biofuels and other solvent-like chemicals, product toxicity is one of the main bottlenecks in achieving optimal production. Since overcoming sensitivity to the final product often requires engineering tolerance mechanisms, this specific area of host optimization has recently been termed tolerance engineering [2Ling H. et al.Microbial tolerance engineering toward biochemical production: from lignocellulose to products.Curr. Opin. Biotechnol. 2014; 29: 99-106Crossref PubMed Scopus (3) Google Scholar, 3Nicolaou S.A. et al.A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation.Metab. Eng. 2010; 12: 307-331Crossref PubMed Scopus (214) Google Scholar, 4Peabody V. et al.Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.Curr. Opin. Chem. Eng. 2014; 6: 9-17Crossref Google Scholar]. Many compounds of industrial value are solvent-like hydrocarbons and range from polymer and plastic precursors to fuels. In the past decade, production of an impressive array of solvent-like compounds has been demonstrated in microbial hosts (Table 1) [1Alper H. Stephanopoulos G. Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?.Nat. Rev. Microbiol. 2009; 7: 715-723Crossref PubMed Scopus (184) Google Scholar, 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholar, 10Fortman J.L. et al.Biofuel alternatives to ethanol: pumping the microbial well.Trends Biotechnol. 2008; 26: 375-381Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar]. The motivation underlying the development of sustainable and biological routes to manufacture these compounds is a densely and thoroughly reviewed topic [1Alper H. Stephanopoulos G. Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?.Nat. Rev. Microbiol. 2009; 7: 715-723Crossref PubMed Scopus (184) Google Scholar, 7Erickson B. et al.Perspective on opportunities in industrial biotechnology in renewable chemicals.Biotechnol. J. 2012; 7: 176-185Crossref PubMed Scopus (61) Google Scholar, 10Fortman J.L. et al.Biofuel alternatives to ethanol: pumping the microbial well.Trends Biotechnol. 2008; 26: 375-381Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar, 12Papoutsakis E.T. Engineering solventogenic clostridia.Curr. Opin. Biotechnol. 2008; 19: 420-429Crossref PubMed Scopus (180) Google Scholar]. It is worth noting that microbial production of these compounds is distinguished from the production of fine chemicals by the eventual scale at which the process will have to be implemented to be economically viable. Further, many of these compounds negatively impact the growth of microbial production strains. The past decade has seen an abundance of efforts in understanding the cause and mechanism of solvent stress. Moving forward, the field must focus on how to apply these discoveries to alleviate the sensitivity and improve microbial robustness. Here we briefly review the present knowledge around solvent toxicity and notable recent advances in developing solvent-tolerant bacterial strains, and focus on gene targets that have led to improvement in production. In particular, we review the role of transporters in tolerance and strain engineering.Table 1Representative solvent-like fuels and chemicalsCompoundType of useHost strainTypical recent productionaValues listed do not take into account differences such as medium used, glucose levels, aeration, cultivation mode (batch, fed batch, or continuous), scale, and time of measurement., bFor additional production parameters and additional organic acids, diacids, alcohols, and diols, see [9,23]. (g/l)Log PcLog P values (Box 1) obtained from the miLogP site (http://www.molinspiration.com/services/logp.html).ToxicityaValues listed do not take into account differences such as medium used, glucose levels, aeration, cultivation mode (batch, fed batch, or continuous), scale, and time of measurement., dToxicity is reported using very different assays in the literature [15,20,23,65,75,86,101,102]: % values represent v/v levels sufficient to cause decrease in growth by at least half of optimal growth in the given media. (% v/v)C4n-ButanolFuel, solventEscherichia coliClostridium acetobutylicumPseudomonas putida30 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar5–19 8Ezeji T. et al.Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms.Appl. Microbiol. Biotechnol. 2010; 85: 1697-1712Crossref PubMed Scopus (125) Google Scholar, 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholar, 103Jang Y.S. et al.Butanol production from renewable biomass: rediscovery of metabolic pathways and metabolic engineering.Biotechnol. J. 2012; 7: 186-198Crossref PubMed Scopus (48) Google Scholar0.12 104Nielsen D.R. et al.Engineering alternative butanol production platforms in heterologous bacteria.Metab. Eng. 2009; 11: 262-273Crossref PubMed Scopus (191) Google Scholar1.121.5%1.6%1.5%IsobutanolFuel, solventE. coli50 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar0.800.8%1,4-ButanediolPolymer precursorE. coli18 6Yim H. et al.Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.Nat. Chem. Biol. 2011; 7: 445-452Crossref PubMed Scopus (289) Google Scholar−0.412%C5n-PentanolFuel, solventE. coli0.486 98Tseng H.C. Prather K.L. Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 17925-17930Crossref PubMed Scopus (33) Google Scholar2.2 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar1.620.1%3-Methyl 1-butenolFuel, solventE. coli4.4–9.5 60Connor M.R. et al.3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation.Appl. Microbiol. Biotechnol. 2010; 86: 1155-1164Crossref PubMed Scopus (78) Google Scholar0.1%3-Methyl 3-butenolFuel, solventE. coli1.5 84George K.W. et al.Correlation analysis of targeted proteins and metabolites to assess and engineer microbial isopentenol production.Biotechnol. Bioeng. 2014; 111: 1648-1658Crossref PubMed Scopus (2) Google Scholar1.360.1%IsopentanolFuel, solventE. coli2.2 105Connor M.R. Liao J.C. Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol.Appl. Environ. Microbiol. 2008; 74: 5769-5775Crossref PubMed Scopus (72) Google Scholar2.570.1%C6/7n-HexaneSolventE. colin.r.3.66104-fold decrease in CFUs1-HexanolSolventE. coli0.047 11Lamsen E.N. Atsumi S. Recent progress in synthetic biology for microbial production of C3-C10 alcohols.Front. Microbiol. 2012; 3: 196Crossref PubMed Scopus (14) Google Scholar3.147.8 × 10−3 mM1-HexenePolymer precursorE. colin.r.3.1470 mg/LTolueneFuel additiveE. coliP. putidan.r.n.r.2.390.04%0.3%C8StyrenePolymer precursorE. coli0.260 63McKenna R. et al.Comparing in situ removal strategies for improving styrene bioproduction.Bioprocess Biosyst. Eng. 2015; 38: 165-174Crossref PubMed Scopus (1) Google Scholar, 106McKenna R. Nielsen D.R. Styrene biosynthesis from glucose by engineered E. coli.Metab. Eng. 2011; 13: 544-554Crossref PubMed Scopus (44) Google Scholar2.79100 mg/Lp-Hydroxy styrenePolymer precursorE. coliP. putida S123.3 mM 102Ben-Bassat, A. et al. E.I. Du Pont De Nemours. Microbial conversion of glucose to para-hydroxystyrene, US7229806 B2Google Scholar4.5 mM 62Verhoef S. et al.Bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium Pseudomonas putida S12 in a two-phase water–decanol fermentation.Appl. Environ. Microbiol. 2009; 75: 931-936Crossref PubMed Scopus (40) Google Scholar0.5 g/ltolerant1-OctanolBiodiesel, solventE. coli0.015–0.1 77Akhtar M.K. et al.Microbial production of 1-octanol: a naturally excreted biofuel with diesel-like properties.Metab. Eng. Commun. 2015; 2: 1-5Crossref Google Scholar3.140.025%Adipic acidPolymer precursorE. coli0.64 mg/l 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholar0.36n.r.C10GeraniolFuel–n.r.3.200.05%LimoneneFuel, disinfectantE. coliSynechococcus sp. PCC 70020.4 97Alonso-Gutierrez J. et al.Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.Metab. Eng. 2013; 19: 33-41Crossref PubMed Scopus (30) Google Scholar0.4 mg/l 107Davies F.K. et al.Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002.Front. Bioeng. Biotechnol. 2014; 2: 21Crossref Google Scholar3.610.025%eAcute limonene toxicity is caused by products of limonene oxidation such as limonene hydroperoxide [132].n.r.Alpha-pineneFuel, disinfectantE. coli0.028 108Sarria S. et al.Microbial synthesis of pinene.ACS Synth. Biol. 2014; 3: 466-475Crossref PubMed Scopus (10) Google Scholar3.540.5%≥C15BisaboleneFuelE. coliSynechococcus sp. PCC 70020.9 109Peralta-Yahya P.P. et al.Identification and microbial production of a terpene-based advanced biofuel.Nat. Commun. 2011; 2: 483Crossref PubMed Scopus (151) Google Scholar0.6 mg/l 107Davies F.K. et al.Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002.Front. Bioeng. Biotechnol. 2014; 2: 21Crossref Google Scholar5.36n.d.n.r.FFA (C8–C12)FuelE. coli0.8–1 110Lennen R.M. Pfleger B.F. Engineering Escherichia coli to synthesize free fatty acids.Trends Biotechnol. 2012; 30: 659-667Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 111Sherkhanov S. et al.Improving the tolerance of Escherichia coli to medium-chain fatty acid production.Metab. Eng. 2014; 25: 1-7Crossref PubMed Scopus (1) Google Scholarn.a.Tenfold decrease in CFUsFatty acid ethyl esters (FAEEs)FuelE. coli0.580 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholarn.a.n.d.CFU, colony-forming unit; n.d., none detected; n.r., not reported; n.a., not applicable.a Values listed do not take into account differences such as medium used, glucose levels, aeration, cultivation mode (batch, fed batch, or continuous), scale, and time of measurement.b For additional production parameters and additional organic acids, diacids, alcohols, and diols, see 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholar, 23Huffer S. et al.Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.Appl. Environ. Microbiol. 2011; 77: 6400-6408Crossref PubMed Scopus (38) Google Scholar.c Log P values (Box 1) obtained from the miLogP site (http://www.molinspiration.com/services/logp.html).d Toxicity is reported using very different assays in the literature 15Segura A. et al.Solvent tolerance in Gram-negative bacteria.Curr. Opin. Biotechnol. 2012; 23: 415-421Crossref PubMed Scopus (26) Google Scholar, 20Lennen R.M. et al.Identification of transport proteins involved in free fatty acid efflux in Escherichia coli.J. Bacteriol. 2013; 195: 135-144Crossref PubMed Scopus (19) Google Scholar, 23Huffer S. et al.Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.Appl. Environ. Microbiol. 2011; 77: 6400-6408Crossref PubMed Scopus (38) Google Scholar, 65Dunlop M.J. et al.Engineering microbial biofuel tolerance and export using efflux pumps.Mol. Syst. Biol. 2011; 7: 487Crossref PubMed Scopus (163) Google Scholar, 75Mingardon F. et al.Improving olefin tolerance and production in E. coli using native and evolved AcrB.Biotechnol. Bioeng. 2015; 112: 879-888Crossref PubMed Google Scholar, 86Doukyu N. et al.Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli.J. Appl. Microbiol. 2012; 112: 464-474Crossref PubMed Scopus (6) Google Scholar, 101Tsai J.T. et al.Production of toluene cis-glycol by a recombinant Escherichia coli strain in a two-liquid phase culture system.Biotechnol. Lett. 1996; 18: 241-244Crossref Google Scholar, 102Ben-Bassat, A. et al. E.I. Du Pont De Nemours. Microbial conversion of glucose to para-hydroxystyrene, US7229806 B2Google Scholar: % values represent v/v levels sufficient to cause decrease in growth by at least half of optimal growth in the given media.e Acute limonene toxicity is caused by products of limonene oxidation such as limonene hydroperoxide 132Chubukov V. et al.Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC).Appl. Environ. Microbiol. 2015; (Published online May 1, 2015)https://doi.org/10.1128/AEM.01102-15Crossref PubMed Scopus (3) Google Scholar. Open table in a new tab CFU, colony-forming unit; n.d., none detected; n.r., not reported; n.a., not applicable. Microbial solvent tolerance is not a new area of research. This topic has been explored from the viewpoint of bioremediation of hydrocarbon spills and contamination, biocatalysis using whole microbes in two-phase solvent systems, and the production of solvent-like compounds. Several recent and thoughtful reviews [3Nicolaou S.A. et al.A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation.Metab. Eng. 2010; 12: 307-331Crossref PubMed Scopus (214) Google Scholar, 4Peabody V. et al.Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.Curr. Opin. Chem. Eng. 2014; 6: 9-17Crossref Google Scholar, 9Cho C. et al.Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.Biotechnol. Adv. 2014; (Published online November 18, 2014)https://doi.org/10.1016/j.biotechadv.2014.11.006Crossref PubMed Scopus (3) Google Scholar, 13Kell D.B. et al.Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.Trends Biotechnol. 2015; 33: 237-246Abstract Full Text Full Text PDF PubMed Google Scholar, 14Dunlop M.J. Engineering microbes for tolerance to next-generation biofuels.Biotechnol. Biofuels. 2011; 4: 32Crossref PubMed Scopus (89) Google Scholar, 15Segura A. et al.Solvent tolerance in Gram-negative bacteria.Curr. Opin. Biotechnol. 2012; 23: 415-421Crossref PubMed Scopus (26) Google Scholar, 16Jin H. et al.Engineering biofuel tolerance in non-native producing microorganisms.Biotechnol. Adv. 2014; 32: 541-548Crossref PubMed Scopus (5) Google Scholar] and comprehensive systems biology studies [17Atsumi S. et al.Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli.Mol. Syst. Biol. 2010; 6: 449Crossref PubMed Scopus (106) Google Scholar, 18Minty J.J. et al.Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.Microb. Cell Fact. 2011; 10: 18Crossref PubMed Scopus (70) Google Scholar, 19Reyes L.H. et al.Genetic determinants for n-butanol tolerance in evolved Escherichia coli mutants: cross adaptation and antagonistic pleiotropy between n-butanol and other stressors.Appl. Environ. Microbiol. 2013; 79: 5313-5320Crossref PubMed Scopus (6) Google Scholar, 20Lennen R.M. et al.Identification of transport proteins involved in free fatty acid efflux in Escherichia coli.J. Bacteriol. 2013; 195: 135-144Crossref PubMed Scopus (19) Google Scholar, 21Anfelt J. et al.Using transcriptomics to improve butanol tolerance of Synechocystis sp. strain PCC 6803.Appl. Environ. Microbiol. 2013; 79: 7419-7427Crossref PubMed Scopus (7) Google Scholar, 22Brynildsen M.P. Liao J.C. An integrated network approach identifies the isobutanol response network of Escherichia coli.Mol. Syst. Biol. 2009; 5: 277Crossref PubMed Scopus (94) Google Scholar, 23Huffer S. et al.Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.Appl. Environ. Microbiol. 2011; 77: 6400-6408Crossref PubMed Scopus (38) Google Scholar, 24Rutherford B.J. et al.Functional genomic study of exogenous n-butanol stress in Escherichia coli.Appl. Environ. Microbiol. 2010; 76: 1935-1945Crossref PubMed Scopus (106) Google Scholar, 25Zingaro K.A. Papoutsakis E.T. Toward a semisynthetic stress response system to engineer microbial solvent tolerance.Mbio. 2012; 3: e00308-e312Crossref Scopus (26) Google Scholar, 26Udaondo Z. et al.Analysis of solvent tolerance in Pseudomonas putida DOT-T1E based on its genome sequence and a collection of mutants.FEBS Lett. 2012; 586: 2932-2938Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 27Ruffing A.M. Trahan C.A. Biofuel toxicity and mechanisms of biofuel tolerance in three model cyanobacteria.Algal Res. 2014; 5: 121-132Crossref Google Scholar] focus on evaluating solvent tolerance across numerous bacterial hosts (also see Box 1). Although a given solvent will elicit specific stress responses, some generalizable trends are now established. Predominantly hydrophobic compounds (e.g., aromatics, alkanes) intercalate into the membrane and alter membrane fluidity; therefore, responses that counteract this perturbation frequently include inner membrane lipid modifications. Cis-to-trans isomerization in fatty acids is documented to occur within minutes of solvent exposure in Pseudomonas strains [28Heipieper H.J. Fischer J. Bacterial solvent responses and tolerance: cis–trans isomerization.in: Timmis K. Handbook of Hydrocarbon and Lipid Microbiology. Springer, 2010: 4203-4211Crossref Google Scholar], while increases in cyclopropyl fatty acids [29Pini C.V. et al.Cyclopropane fatty acids are involved in organic solvent tolerance but not in acid stress resistance in Pseudomonas putida DOT-T1E.Microb. Biotechnol. 2009; 2: 253-261Crossref PubMed Scopus (16) Google Scholar, 30Pinkart H.C. et al.Cell envelope changes in solvent-tolerant and solvent-sensitive Pseudomonas putida strains following exposure to o-xylene.Appl. Environ. Microbiol. 1996; 62: 1129-1132Crossref PubMed Google Scholar] or changes in fatty acid chain length [31Ingram L.O. Adaptation of membrane lipids to alcohols.J. Bacteriol. 1976; 125: 670-678Crossref PubMed Google Scholar, 32Ramos J.L. et al.Mechanisms of solvent tolerance in Gram-negative bacteria.Annu. Rev. Microbiol. 2002; 56: 743-768Crossref PubMed Scopus (437) Google Scholar, 33Royce L.A. et al.The damaging effects of short chain fatty acids on Escherichia coli membranes.Appl. Microbiol. Biotechnol. 2013; 97: 8317-8327Crossref PubMed Scopus (10) Google Scholar] are also reported and occur at later time points. Subsequently, pumps are induced to export the product and minimize the solvent presence within the cell (e.g., AcrAB-TolC, TtgABC, SrpABC [15Segura A. et al.Solvent tolerance in Gram-negative bacteria.Curr. Opin. Biotechnol. 2012; 23: 415-421Crossref PubMed Scopus (26) Google Scholar, 32Ramos J.L. et al.Mechanisms of solvent tolerance in Gram-negative bacteria.Annu. Rev. Microbiol. 2002; 56: 743-768Crossref PubMed Scopus (437) Google Scholar, 34Segura A. Ramos J. Toluene tolerance systems in Pseudomonas.in: Nojiri H. Biodegradative Bacteria. Springer, 2014: 227-248Crossref Google Scholar]). Non-optimal membrane fluidity results in disruption of membrane-bound complexes, adversely affecting energy-generating complexes (TonB, ion and H+ transport, and ATP synthesis [32Ramos J.L. et al.Mechanisms of solvent tolerance in Gram-negative bacteria.Annu. Rev. Microbiol. 2002; 56: 743-768Crossref PubMed Scopus (437) Google Scholar]), metabolite loss [23Huffer S. et al.Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.Appl. Environ. Microbiol. 2011; 77: 6400-6408Crossref PubMed Scopus (38) Google Scholar], and general extracytoplasmic stress [24Rutherford B.J. et al.Functional genomic study of exogenous n-butanol stress in Escherichia coli.Appl. Environ. Microbiol. 2010; 76: 1935-1945Crossref PubMed Scopus (106) Google Scholar], comprehensively reviewed by Nicolaou et al. [3Nicolaou S.A. et al.A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation.Metab. Eng. 2010; 12: 307-331Crossref PubMed Scopus (214) Google Scholar]. In response to protein misfolding and damage, upregulation in chaperones and degradative complexes (e.g., GroESL, ClpB) are often observed during solvent exposure [2Ling H. et al.Microbial tolerance engineering toward biochemical production: from lignocellulose to products.Curr. Opin. Biotechnol. 2014; 29: 99-106Crossref PubMed Scopus (3) Google Scholar, 3Nicolaou S.A. et al.A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation.Metab. Eng. 2010; 12: 307-331Crossref PubMed Scopus (214) Google Scholar, 4Peabody V. et al.Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.Curr. Opin. Chem. Eng. 2014; 6: 9-17Crossref Google Scholar, 19Reyes L.H. et al.Genetic determinants for n-butanol tolerance in evolved Escherichia coli mutants: cross adaptation and antagonistic pleiotropy between n-butanol and other stressors.Appl. Environ. Microbiol. 2013; 79: 5313-5320Crossref PubMed Scopus (6) Google Scholar, 35Papoutsakis E. Alsaker K. Towards a synthetic biology of the stress-response and the tolerance phenotype: systems understanding and engineering of the Clostridium acetobutylicum stress-response and tolerance to toxic metabolites.in: Wittmann C. Lee S.Y. Systems Metabolic Engineering. Springer, 2012: 193-219Crossref Google Scholar]. This is an especially strong component of the stress response for solvents with polar groups (e.g., short-chain alcohols) [18Minty J.J. et al.Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.Microb. Cell Fact. 2011; 10: 18Crossref PubMed Scopus (70) Google Scholar, 19Reyes L.H. et al.Genetic determinants for n-butanol tolerance in evolved Escherichia coli mutants: cross adaptation and antagonistic pleiotropy between n-butanol and other stressors.Appl. Environ. Microbiol. 2013; 79: 5313-5320Crossref PubMed Scopus (6) Google Scholar, 23Huffer S. et al.Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.Appl. Environ. Microbiol. 2011; 77: 6400-6408Crossref PubMed Scopus (38) Google Scholar, 35Papoutsakis E. Alsaker K. Towards a synthetic biology of the stress-response and the tolerance phenotype: systems understanding and engineering of the Clostridium acetobutylicum stress-response and tolerance to toxic metabolites.in: Wittmann C. Lee S.Y. Systems Metabolic Engineering. Springer, 2012: 193-219Crossref Google Scholar], which have greater solubility in the media and access into the cell. Several studies also report the generation of reactive oxygen species (ROS) during exposure to short-chain alc
Referência(s)