臺灣博碩士論文加值系統

在生質燃料中，丁醇是一個相當有潛力的替代性能源。主要都是透過Clostridial 屬的菌株(例如：Clostridium acetobutylicum)經由傳統的ABE (acetone-butanol-ethanol) 醱酵生產。對於傳統的Clostridium丁醇生產者或基因工程改造的Escherichia coli 丁醇生產菌株，其丁醇本身對細胞具有較高的毒性，然而丁醇的耐受性對於生質丁醇的產量亦是瓶頸之一。近年來發現生長在含有丁醇環境下的微生物會導致細胞體內的活性氧化物質增加。然而cysteine 相當豐富的metallothioneins (MTs) 被報導可以清除此類的活性氧化物質，因此本研究透過基因重組的Escherichia coli 將人類 (HMT) 、老鼠(MMT) 和吳郭魚 (TMT) 的MT個別表現於膜內或透過outer membrane protein C (OmpC) 的融合將MT表現於膜上，希望藉由MT清除此類的活性氧化物後進而提升微生物對丁醇的耐受性。結果中顯示，當不同種物種的MT表現於E. coli 的胞內和膜上時，其對E. coli 所提升的丁醇耐受性皆不同，且相較於乙醇，丁醇對細胞的毒害確實較高(對造組於1%的丁醇下即無法存活)。其中表現在膜上的OmpC-TMT組合最佳，可將E. coli 對丁醇的耐受度提升至1.5%~2%，相較於對造組其耐受性提升了約二倍。有鑒於此，本研究透過合成生物學平台的運用以OGAB method (Ordered Gene Assembly in Bacillus subtilis)，將C. acetobutylicum ATCC 824 之產丁醇途徑 (thl、hbd、crt-bcd-etfB-etfA-hbd、adhe) 與membrane-targeted TMT同步構築表現於E .coli 。以單一菌株完成提升丁醇耐受性與產量之醱酵反應程序。結果顯示，透過此策略可以改善菌株細胞膜的完整性並且將丁醇產量由95.1 mg/L 提升至320 mg/L。此外在轉錄分析的結果中發現，有三個主要的KEGG pathways是顯著差異，包含oxidative phosphorylation，fructose and mannose metabolism和glycolysis/gluconeogenesis。由結果中我們推測轉殖株因為較高的丁醇產量導致quinone 的功能喪失，以至於與氧化磷酸化相關的 nuo operon 被負調控，而此一結果會降低降低將NADH轉化為NAD +並產生質子動力的能力。因此fructose and mannose metabolism相關基因與glycolysis/gluconeogenesis相關基因分別被正調控與負調控，藉此調節NADH / NAD +的氧化還原反應並防止額外的ATP被消耗。這些結果也暗指在丁醇合成途徑中需要更多的NADH和ATP。我們的研究證實，透過減少氧化壓力產生，可以增加微生物的膜完整性並且提升有毒化學物質的生產潛力。

n-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. In this research, host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural (HMF), and nickel. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism’s growth rate. Combined with the above advantages, the strain BUT1-DE containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The BUT3-DE improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis. These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

摘要 i
Abstract iii
Chapter I: Introduction 1
1.1. Second-generation biofuel (advanced biofuel) 1
1.2. Studies of n-butanol production in C. acetobutylicum 2
1.3. Heterologous production of butanol in non-clostridial microbes 4
1.4. Problems with the microbial tolerance of butanol 7
1.5. Engineering E. coli to improve butanol tolerance 8
Chapter II: Material and Methods 12
2.1. Sources of bacterium and plasmids 12
2.2. Culture conditions 12
2.2.1. Culture medium 12
2.2.2. Growth conditions 13
2.2.3. Determination of bacterium growth 14
2.3. DNA manipulation and construction of recombinant strains 14
2.3.1. Extraction of genomic DNA of bacteria 14
2.3.2. Plasmids extraction 15
2.3.3. Agarose gel electrophoresis 15
2.3.4. Extract DNA fragments from agarose gel 16
2.3.5. Polymerase chain reaction (PCR) 16
2.3.6. Restriction enzyme digestion and DNA fragments ligation 16
2.3.7. Plasmids and transgenic strains construction 16
2.3.8. Transformation 18
2.4. RNA manipulation 22
2.4.1. RNA extraction 22
2.4.2. Real-time reverse transcriptase PCR 22
2.5. Tolerance assay of toxins 22
2.6. Reactive oxygen species assay by carboxy-H2DCFDA 23
2.7. Analysis of fermentation products 23
2.8. Staining of bacterial suspensions with DAPI and SYTOX Green 24
2.9. Next-generation sequencing and analysis 24
Chapter III: Results and Discussions 26
3-1 Improvement of n-butanol tolerance in E. coli by membrane-targeted tilapia metallothionein 26
3-1.1 Alcohols tolerance assay 26
3-1.2 Measurement of free radical scavenging ability for MTs strains 30
3-1.3 The roles of outer membrane (OM) proteins 32
3-1.4 Tolerance assay of lignocellulose pretreatment’s toxins 34
3-2 Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in E. coli 39
3-2.1. The OGAB of technical concept 39
3-2.2. Construction of butanologenic E. coli strains 39
3-2.3. Growth profile and n-butanol production 43
3-2.4. Improved tolerance and n-butanol production 46
3-2.5. Cell membrane integrity test 49
3-2.6. Free radical scavenging ability for butanologenic E. coli strains 51
3-2.7. Expression profiles of n-butanol and tolerance genes in E. coli 53
3-2.8. Transcriptomic analysis in engineered E. coli 54
Chapter IV: Conclusion 59
Reference 60
Supplementary Material 73
Additional file 1. Primes used for OGAB method (Table S1) 73
Additional file 2. Primes used for qPCR (Table S2) 74
Additional file 3. List of the 147 differentially expressed genes (Table S3) 75

1.Harvey BG, Meylemans HA: The role of butanol in the development of sustainable fuel technologies. Journal of Chemical Technology and Biotechnology 2011, 86:2-9.
2.Procentese A, Raganati F, Olivieri G, Russo ME, de la Feld M, Marzocchella A: Renewable feedstocks for biobutanol production by fermentation. N Biotechnol 2016.
3.Green EM: Fermentative production of butanol - the industrial perspective. Current Opinion in Biotechnology 2011, 22:337-343.
4.Ezeji TC, Qureshi N, Blaschek HP: Bioproduction of butanol from biomass: from genes to bioreactors. Current Opinion in Biotechnology 2007, 18:220-227.
5.Lin YL, Blaschek HP: Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth. Applied and Environmental Microbiology 1983, 45:966-973.
6.Keis S, Bennett CF, Ward VK, Jones DT: Taxonomy and Phylogeny of Industrial Solvent-Producing Clostridia. Int J Syst Bacteriol 1995, 45:693-705.
7.Gottwald M, Hippe H, Gottschalk G: Formation of n-Butanol from d-Glucose by Strains of the "Clostridium tetanomorphum" Group. Appl Environ Microbiol 1984, 48:573-576.
8.George HA, Johnson JL, Moore WEC, Holdeman LV, Chen JS: Acetone, Isopropanol, and Butanol Production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum. Applied and Environmental Microbiology 1983, 45:1160-1163.
9.Yan RT, Zhu CX, Golemboski C, Chen JS: Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum"). Applied and Environmental Microbiology 1988, 54:642-648.
10.Dabrock B, Bahl H, Gottschalk G: Parameters Affecting Solvent Production by Clostridium pasteurianum. Applied and Environmental Microbiology 1992, 58:1233-1239.
11.Vasconcelos I, Girbal L, Soucaille P: Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. Journal of Bacteriology 1994, 176:1443-1450.
12.Bennett GN, Rudolph FB: The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum. Fems Microbiology Reviews 1995, 17:241-249.
13.Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC: Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 2008, 10:305-311.
14.Zheng YN, Li LZ, Xian M, Ma YJ, Yang JM, Xu X, He DZ: Problems with the microbial production of butanol. J Ind Microbiol Biot 2009, 36:1127-1138.
15.Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng QD, Gibson R, Lee HM, Dubois J, Qiu DY, Hitti J, et al: Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. Journal of Bacteriology 2001, 183:4823-4838.
16.Jang YS, Lee J, Malaviya A, Seung DY, Cho JH, Lee SY: Butanol production from renewable biomass: Rediscovery of metabolic pathways and metabolic engineering. Biotechnology Journal 2012, 7:186-198.
17.Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD: Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microbial Cell Factories 2008, 7.
18.Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KLJ: Engineering alternative butanol production platforms in heterologous bacteria. Metabolic Engineering 2009, 11:262-273.
19.Berezina OV, Zakharova NV, Brandt A, Yarotsky SV, Schwarz WH, Zverlov VV: Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Applied Microbiology and Biotechnology 2010, 87:635-646.
20.Murphy CD: The microbial cell factory. Organic & Biomolecular Chemistry 2012, 10:1949-1957.
21.Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H: Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Applied Microbiology and Biotechnology 2008, 77:1305-1316.
22.Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R: Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 2011, 476:355-U131.
23.Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC: Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli. Applied and Environmental Microbiology 2011, 77:2905-2915.
24.Ye Q, Bao J, Zhong JJ: Bioreactor Engineering Research and Industrial Applications I Cell Factories Preface. Adv Biochem Eng Biot 2016, 155:V-Vii.
25.Bowles LK, Ellefson WL: Effects of butanol on Clostridium acetobutylicum. Appl Environ Microbiol 1985, 50:1165-1170.
26.Knoshaug EP, Zhang M: Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 2009, 153:13-20.
27.Mao S, Luo Y, Zhang T, Li J, Bao G, Zhu Y, Chen Z, Zhang Y, Li Y, Ma Y: Proteome reference map and comparative proteomic analysis between a wild type Clostridium acetobutylicum DSM 1731 and its mutant with enhanced butanol tolerance and butanol yield. J Proteome Res 2010, 9:3046-3061.
28.Liu XB, Gu QY, Yu XB: Repetitive domestication to enhance butanol tolerance and production in Clostridium acetobutylicum through artificial simulation of bio-evolution. Bioresour Technol 2013, 130:638-643.
29.Tomas CA, Beamish J, Papoutsakis ET: Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol 2004, 186:2006-2018.
30.Rutherford BJ, Dahl RH, Price RE, Szmidt HL, Benke PI, Mukhopadhyay A, Keasling JD: Functional genomic study of exogenous n-butanol stress in Escherichia coli. Appl Environ Microbiol 2010, 76:1935-1945.
31.Brynildsen MP, Liao JC: An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol 2009, 5:277.
32.Gonzalez R, Tao H, Purvis JE, York SW, Shanmugam KT, Ingram LO: Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol Prog 2003, 19:612-623.
33.Sigler K, Chaloupka J, Brozmanova J, Stadler N, Hofer M: Oxidative stress in microorganisms-I. Microbial vs. higher cells-damage and defenses in relation to cell aging and death. Folia Microbiol (Praha) 1999, 44:587-624.
34.Jones DP: Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 2008, 295:C849-868.
35.Perrone GG, Tan SX, Dawes IW: Reactive oxygen species and yeast apoptosis. Biochim Biophys Acta 2008, 1783:1354-1368.
36.Fridovich I: Superoxide radical and superoxide dismutases. Annual review of biochemistry 1995, 64:97-112.
37.Imlay JA: Pathways of oxidative damage. Annu Rev Microbiol 2003, 57:395-418.
38.Coyle P, Philcox JC, Carey LC, Rofe AM: Metallothionein: the multipurpose protein. Cell Mol Life Sci 2002, 59:627-647.
39.Vallee BL: The function of metallothionein. Neurochem Int 1995, 27:23-33.
40.Park JD, Liu Y, Klaassen CD: Protective effect of metallothionein against the toxicity of cadmium and other metals(1). Toxicology 2001, 163:93-100.
41.Sato M, Kondoh M: Recent studies on metallothionein: protection against toxicity of heavy metals and oxygen free radicals. Tohoku J Exp Med 2002, 196:9-22.
42.Lin KH, Chien MF, Hsieh JL, Huang CC: Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein. Appl Microbiol Biotechnol 2010.
43.Puente JL, Juarez D, Bobadilla M, Arias CF, Calva E: The Salmonella ompC gene: structure and use as a carrier for heterologous sequences. Gene 1995, 156:1-9.
44.Kempf B, Bremer E: Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol 1998, 170:319-330.
45.Xu Z, Lee SY: Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl Environ Microbiol 1999, 65:5142-5147.
46.Tsuge K, Matsui K, Itaya M: One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res 2003, 31:e133.
47.Miller J: Experiments in molecular genetics. 1972.
48.Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. Cold spring harbor laboratory press; 1989.
49.Anagnostopoulos C, Spizizen J: Requirements for Transformation in Bacillus Subtilis. J Bacteriol 1961, 81:741-746.
50.Harwood CR, Cutting SM: Plasmids. In "Molecular biological methods for Bacillus". C. R. Harwood and S. M. Cutting edn: John Wiley and Sons, Chichester, UK; 1990.
51.Borden JR, Papoutsakis ET: Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum. Appl Environ Microbiol 2007, 73:3061-3068.
52.Bolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30:2114-2120.
53.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 2012, 7:562-578.
54.NIH DAVID Bioinformatics Resources 6.7. Available online: http://david.abcc.ncifcrf.gov/ (accessed on 28 December 2014).
55.Ruhl J, Schmid A, Blank LM: Selected Pseudomonas putida strains able to grow in the presence of high butanol concentrations. Appl Environ Microbiol 2009, 75:4653-4656.
56.Dien B, Cotta M, Jeffries T: Bacteria engineered for fuel ethanol production: current status. Applied microbiology and biotechnology 2003, 63:258-266.
57.Casadei, Ingram R, Hitchings E, Archer J, Gaze JE: Heat resistance of Bacillus cereus, Salmonella typhimurium and Lactobacillus delbrueckii in relation to pH and ethanol. Int J Food Microbiol 2001, 63:125-134.
58.Kataoka N, Tajima T, Kato J, Rachadech W, Vangnai AS: Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host. AMB Express 2011, 1:10.
59.Brown SD, Guss AM, Karpinets TV, Parks JM, Smolin N, Yang S, Land ML, Klingeman DM, Bhandiwad A, Rodriguez M, Jr., et al: Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum. Proc Natl Acad Sci U S A 2011, 108:13752-13757.
60.Trautwein K, Kuhner S, Wohlbrand L, Halder T, Kuchta K, Steinbuchel A, Rabus R: Solvent stress response of the denitrifying bacterium "Aromatoleum aromaticum" strain EbN1. Appl Environ Microbiol 2008, 74:2267-2274.
61.Landfald B, Strom AR: Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol 1986, 165:849-855.
62.Yang JN, Wang C, Guo C, Peng XX, Li H: Outer membrane proteome and its regulation networks in response to glucose concentration changes in Escherichia coli. Molecular bioSystems 2011, 7:3087-3093.
63.Ingram LO, Aldrich HC, Borges AC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, et al: Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog 1999, 15:855-866.
64.Almeida JR, Bertilsson M, Gorwa-Grauslund MF, Gorsich S, Liden G: Metabolic effects of furaldehydes and impacts on biotechnological processes. Appl Microbiol Biotechnol 2009, 82:625-638.
65.Mills TY, Sandoval NR, Gill RT: Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnology for biofuels 2009, 2:26.
66.Chin WC, Lin KH, Chang JJ, Huang CC: Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein. Biotechnology for biofuels 2013, 6:130.
67.Nishizaki T, Tsuge K, Itaya M, Doi N, Yanagawa H: Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol 2007, 73:1355-1361.
68.Uozumi T, Hoshino T, Miwa K, Horinouchi S, Beppu T, Arima K: Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I. Mol Gen Genet 1977, 152:65-69.
69.Kaneko S, Akioka M, Tsuge K, Itaya M: DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector. Journal of molecular biology 2005, 349:1036-1044.
70.Zhu L, Dong H, Zhang Y, Li Y: Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability. Metab Eng 2011, 13:426-434.
71.Boyarskiy S, Davis Lopez S, Kong N, Tullman-Ercek D: Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol. Metab Eng 2016, 33:130-137.
72.Zhang H, Chong H, Ching CB, Song H, Jiang R: Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance. Appl Microbiol Biotechnol 2012, 94:1107-1117.
73.Zingaro KA, Terry Papoutsakis E: GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns. Metab Eng 2013, 15:196-205.
74.Watanabe R, Doukyu N: Improvement of organic solvent tolerance by disruption of the lon gene in Escherichia coli. J Biosci Bioeng 2014, 118:139-144.
75.Bui le M, Lee JY, Geraldi A, Rahman Z, Lee JH, Kim SC: Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions. J Biotechnol 2015, 204:33-44.
76.Si HM, Zhang F, Wu AN, Han RZ, Xu GC, Ni Y: DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli. Biotechnology for biofuels 2016, 9:114.
77.Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD: Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010, 463:559-562.
78.Mialon L, Vanderhenst R, Pemba AG, Miller SA: Polyalkylenehydroxybenzoates (PAHBs): biorenewable aromatic/aliphatic polyesters from lignin. Macromolecular rapid communications 2011, 32:1386-1392.
79.Polen T, Spelberg M, Bott M: Toward biotechnological production of adipic acid and precursors from biorenewables. J Biotechnol 2013, 167:75-84.
80.Lennen RM, Kruziki MA, Kumar K, Zinkel RA, Burnum KE, Lipton MS, Hoover SW, Ranatunga DR, Wittkopp TM, Marner WD, 2nd, Pfleger BF: Membrane stresses induced by overproduction of free fatty acids in Escherichia coli. Appl Environ Microbiol 2011, 77:8114-8128.
81.Jarboe LR, Royce LA, Liu P: Understanding biocatalyst inhibition by carboxylic acids. Frontiers in microbiology 2013, 4:272.
82.Lennen RM, Pfleger BF: Modulating membrane composition alters free fatty acid tolerance in Escherichia coli. PLoS One 2013, 8:e54031.
83.Royce LA, Yoon JM, Chen Y, Rickenbach E, Shanks JV, Jarboe LR: Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity. Metab Eng 2015, 29:180-188.
84.Tan Z, Yoon JM, Nielsen DR, Shanks JV, Jarboe LR: Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables. Metabolic engineering 2016, 35:105-113.
85.Ingram LO: Microbial tolerance to alcohols: role of the cell membrane. Trends Biotechnol 1986, 4:40-44.
86.Aono R, Nakajima H: Organic solvent tolerance in Escherichia coli. Tanpakushitsu Kakusan Koso 1997, 42:2532-2541.
87.Ingram LO, Buttke TM: Effects of alcohols on micro-organisms. Adv Microb Physiol 1984, 25:253-300.
88.Kabelitz N, Santos PM, Heipieper HJ: Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus. FEMS Microbiol Lett 2003, 220:223-227.
89.Minty JJ, Lesnefsky AA, Lin F, Chen Y, Zaroff TA, Veloso AB, Xie B, McConnell CA, Ward RJ, Schwartz DR, et al: Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli. Microb Cell Fact 2011, 10:18.
90.Ingram LO: Ethanol tolerance in bacteria. Crit Rev Biotechnol 1990, 9:305-319.
91.Goodarzi H, Bennett BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S: Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli. Mol Syst Biol 2010, 6:378.
92.Woodruff LB, Pandhal J, Ow SY, Karimpour-Fard A, Weiss SJ, Wright PC, Gill RT: Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli. Metab Eng 2013, 15:124-133.
93.Asako H, Nakajima H, Kobayashi K, Kobayashi M, Aono R: Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microbiol 1997, 63:1428-1433.
94.Luo LH, Seo PS, Seo JW, Heo SY, Kim DH, Kim CH: Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation. Biotechnol Lett 2009, 31:1867-1871.
95.Jarboe LR, Liu P, Royce LA: Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals. Current Opinion in Chemical Engineering 2011, 1:38-42.
96.Ingledew WJ, Poole RK: The respiratory chains of Escherichia coli. Microbiol Rev 1984, 48:222-271.
97.Georgellis D, Kwon O, Lin EC: Quinones as the redox signal for the arc two-component system of bacteria. Science 2001, 292:2314-2316.
98.Malpica R, Franco B, Rodriguez C, Kwon O, Georgellis D: Identification of a quinone-sensitive redox switch in the ArcB sensor kinase. Proc Natl Acad Sci U S A 2004, 101:13318-13323.
99.Malpica R, Sandoval GR, Rodriguez C, Franco B, Georgellis D: Signaling by the arc two-component system provides a link between the redox state of the quinone pool and gene expression. Antioxid Redox Signal 2006, 8:781-795.
100.Saini M, Li SY, Wang ZW, Chiang CJ, Chao YP: Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol. Biotechnology for biofuels 2016, 9:69.
101.Du Toit PJ, Kotze JP: The isolation and characterization of sorbitol-6-phosphate dehydrogenase from Clostridium pasteurianum. Biochim Biophys Acta 1970, 206:333-342.
102.Hacking AJ, Lin EC: Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli. J Bacteriol 1976, 126:1166-1172.
103.White D: Physiology and biochemistry of prokaryotes. 2007.
104.Sundara Sekar B, Seol E, Mohan Raj S, Park S: Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: reduction of pyruvate accumulation. Biotechnology for biofuels 2016, 9:95.
105.Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E: The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem 2004, 279:6613-6619.