Abstract
Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l−1 ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l−1) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l−1 and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.
Similar content being viewed by others
References
Buckley M, Wall J (2006) Microbial energy conversion. A report from the American Academy of Microbiology, Washington DC
Burdette DS, Jung SH, Shen GJ, Hollingsworth RI, Zeikus JG (2002) Physiological function of alcohol dehydrogenases and long-chain (C(30)) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus. Appl Environ Microbiol 68(4):1914–1918
Chen K, Iverson AG, Garza EA, Grayburn WS, Zhou S (2010) Metabolic evolution of non-transgenic Escherichia coli SZ420 for enhanced homoethanol fermentation from xylose. Biotechnol Lett 32:87–96
Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63(3):258–266
Gonzalez R, Tao H, Purvis JE, York SW, Shanmugam KT, Ingram LO (2003) 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 19(2):612–623
Hahn-Hagerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953
Ho NMY, Chen Z, Brainard AP (1998) Genetically engineered Saccharomyces yeast capable of effective co-fermentation of glucose and xylose. Appl Environ Microbiol 64:1852–1859
Ingram LO (1981) Mechanism of lysis of Escherichia coli by ethanol and other chaotropic agents. J Bacteriol 146:331–336
Ingram LO (1982) On the regulation of fatty acid composition of Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents and reduction in growth temperature. J Bacteriol 149:166–172
Ingram LO (1989) Ethanol tolerance in bacteria. Crit Rev Biotechnol 9(4):305–319
Ingram LO, Dombek KM (1989) Effects of ethanol on Escherichia coli. In: van Uden N (ed) Alcohol toxicity in yeast and bacteria. CRC, Boca Raton, pp 227–238
Jeffries TW (2006) Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 17(3):320–326
Kuyper M, Hartogg MM, Toirkens MJ, Almering MJ, Winkler AA, van Dijken JP, Pronk JT (2005) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 5(4–5):399–409
Liu S, Qureshi N (2009) How microbes tolerate ethanol and butanol. New Biotechnol 26(3–4):117–121
Lovitt RW, Shen GJ, Zeihus JG (1988) Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum. J Bacteriol 170(6):2809–2815
Luo LH, Seo PS, Seo JW, Heo SY, Kim DH, Kim CH (2009) Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation. Biotechnol Lett 31:1867–1871
Pinkart HC, White DC (1997) Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains. J Bacteriol 179:4219–4226
Sikkema J, de Bont JAM, Poolman B (1995) Mechanism of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222
Song S, Park C (1997) Organization and regulation of the d-xylose operon in Escherichia coli K-12: XylR acts as a transcriptional activator. J Bacteriol 179(22):7025–7032
Sudha Rani K, Swamy MV, Sunitha D, Haritha D, Seenayya G (1996) Improved ethanol tolerance and production in strains of Clostridium thermocellum. World J Microbiol Biotechnol 12(1):57–60
Trinh CT, Huffer S, Clark ME, Blanch HW, Clark DS (2010) Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli. Biotechnol Bioeng. doi:10.1002/bit.22743
Wang Z, Chen M, Xu Y, Li S, Lu W, Ping S, Zhang W, Lin M (2008) An ethanol-tolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose. Biotechnol Lett 30:657–663
Yomano LP, York SW, Ingram LO (1998) Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J Ind Microbiol Biotechnol 20:132–138
Yomano LP, York S, Zhou S, Shanmugam KT, Ingram LO (2008) Re-engineering Escherichia coli for ethanol production. Biotechnol Lett 30(12):2097–2103
Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S (1995) Metabolic engineering a pentose pathway in ethanologenic Zymomonas mobilis. Science 267:240–243
Zhou S, Iverson AG, Grayburn WS (2008) Engineering a native homoethanol pathway in Escherichia coli B for ethanol production. Biotechnol Lett 30:335–342
Zhou S, Iverson AG, Grayburn WS (2010) Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products. Biotechnol Prog 26(1):45–51
Acknowledgments
This research was supported by the Summer Artistry and Research grant, the PMBC research incentive fund of Northern Illinois University, and the China NSF grant (31070094).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wang, Y., Manow, R., Finan, C. et al. Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose. J Ind Microbiol Biotechnol 38, 1371–1377 (2011). https://doi.org/10.1007/s10295-010-0920-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-010-0920-5