Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein

Biotechnology for Biofuels

Chieh-Chen Huang¹  Jui-Jen Chang³  Kuo-Hsing Lin⁴  Wei-Chih Chin² 

[1]Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan

[2]Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan

[3]Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan

[4]Vaccine Research and Development Center, National Institute of Infectious Disease and Vaccinology, NHRI, Miaoli, Taiwan

关键词: Oxidative stress;    E. coli;    Tilapia;    OmpC;    n-butanol;    Metallothionein;   

Others  :  797909
DOI  :  10.1186/1754-6834-6-130

 received in 2013-05-09, accepted in 2013-09-04,  发布年份 2013

【 摘 要 】

Background

Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production.

Results

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. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. 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.

Conclusions

The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.

【 授权许可】

   
2013 Chin et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Ezeji TC, Qureshi N, Blaschek HP: Bioproduction of butanol from biomass: from genes to bioreactors. Curr Opin Biotechnol 2007, 18:220-227.
• [2]Zverlov VV, Berezina O, Velikodvorskaya GA, Schwarz WH: Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery. Appl Microbiol Biotechnol 2006, 71:587-597.
• [3]Bowles LK, Ellefson WL: Effects of butanol on Clostridium acetobutylicum. Appl Environ Microbiol 1985, 50:1165-1170.
• [4]Knoshaug EP, Zhang M: Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 2009, 153:13-20.
• [5]Isaac DD, Pinkner JS, Hultgren SJ, Silhavy TJ: The extracytoplasmic adaptor protein CpxP is degraded with substrate by DegP. Proc Natl Acad Sci USA 2005, 102:17775-17779.
• [6]Ingram LO: Microbial tolerance to alcohols: role of the cell membrane. Trends Biotechnol 1986, 4:40-44.
• [7]Aono R, Nakajima H: [Organic solvent tolerance in Escherichia coli]. Tanpakushitsu Kakusan Koso 1997, 42:2532-2541.
• [8]Ingram LO, Buttke TM: Effects of alcohols on micro-organisms. Adv Microb Physiol 1985, 25:253-300.
• [9]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.
• [10]Ingram LO: Ethanol tolerance in bacteria. Crit Rev Biotechnol 1989, 9:305-319.
• [11]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.
• [12]Tomas CA, Beamish J, Papoutsakis ET: Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum. J Bacteriol 2004, 186:2006-2018.
• [13]Brynildsen MP, Liao JC: An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol 2009, 5:277.
• [14]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.
• [15]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.
• [16]Atsumi S, Wu TY, Machado IM, Huang WC, Chen PY, Pellegrini M, Liao JC: Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol Syst Biol 2010, 6:449.
• [17]Jones DP: Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 2008, 295:C849-C868.
• [18]Imlay JA: Pathways of oxidative damage. Annu Rev Microbiol 2003, 57:395-418.
• [19]Fridovich I: Superoxide radical and superoxide dismutases. Annu Rev Biochem 1995, 64:97-112.
• [20]Coyle P, Philcox JC, Carey LC, Rofe AM: Metallothionein: the multipurpose protein. Cell Mol Life Sci 2002, 59:627-647.
• [21]Vallee BL: The function of metallothionein. Neurochem Int 1995, 27:23-33.
• [22]Park JD, Liu Y, Klaassen CD: Protective effect of metallothionein against the toxicity of cadmium and other metals(1). Toxicology 2001, 163:93-100.
• [23]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.
• [24]Chan KM: Metallothionein: Potential biomarker for monitoring heavy metal pollution in fish around Hong Kong. Mar Pollut Bull 1995, 31:411-415.
• [25]Wong CK, Yeung HY, Cheung RY, Yung KK, Wong MH: Ecotoxicological assessment of persistent organic and heavy metal contamination in Hong Kong coastal sediment. Arch Environ Contam Toxicol 2000, 38:486-493.
• [26]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, 87:561-569.
• [27]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.
• [28]Dien B, Cotta M, Jeffries T: Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003, 63:258-266.
• [29]Casadei MA, 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.
• [30]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. BioMed Central Full Text
• [31]Lin YL, Blaschek HP: Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth. Appl Environ Microbiol 1983, 45:966-973.
• [32]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.
• [33]Perrone GG, Tan SX, Dawes IW: Reactive oxygen species and yeast apoptosis. Biochim Biophys Acta 2008, 1783:1354-1368.
• [34]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.
• [35]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.
• [36]Kempf B, Bremer E: Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol 1998, 170:319-330.
• [37]Landfald B, Strom AR: Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol 1986, 165:849-855.
• [38]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. Mol Biosyst 2011, 7:3087-3093.
• [39]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.
• [40]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.
• [41]Mills TY, Sandoval NR, Gill RT: Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnology for biofuels 2009, 2:26. BioMed Central Full Text
• [42]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.