Genomics on Pretreatment Inhibitor Tolerance of Zymomonas mobilis

Part of the Microbiology Monographs book series (MICROMONO, volume 22)


The development and use of robust ethanologenic microorganisms resistant to industrially relevant pretreatment inhibitors will be a critical component in the successful generation of biofuel on the industrial scale. Recent progress to understand the genetic basis of pretreatment inhibitor tolerance using genomics and systems biology tools for metabolic engineering for the model ethanologenic bacterium Zymomonas mobilis is reviewed in this chapter. The importance of accurate genome annotations and the integration of systems biology data for annotation improvement are highlighted, and case studies that describe the identification and characterization of the Z. mobilis nhaA, hfq, and himA inhibitor tolerance related gene targets are presented.


Zymomonas Mobilis Final Cell Density Comparative Genome Sequencing Inhibitor Tolerance System Biology Study 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank Meghan M. Drake for her careful review and suggestions. The BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the US Department of Energy.


  1. Almeida JRM, Modig T, Petersson A, Hahn-Hagerdal B, Liden G, Gorwa-Grauslund MF (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 82:340–349CrossRefGoogle Scholar
  2. Alper H, Stephanopoulos G (2007) Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab Eng 9:258–267PubMedCrossRefGoogle Scholar
  3. Alper H, Stephanopoulos G (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat Rev Micro 7:715–723CrossRefGoogle Scholar
  4. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314:1565–1568PubMedCrossRefGoogle Scholar
  5. Armengaud J (2009) A perfect genome annotation is within reach with the proteomics and genomics alliance. Curr Opin Microbiol 12:292–300PubMedCrossRefGoogle Scholar
  6. Baudet M, Ortet P, Gaillard JC, Fernandez B, Guerin P, Enjalbal C, Subra G, de Groot A, Barakat M, Dedieu A, Armengaud J (2010) Proteomics-based refinement of Deinococcus deserti genome annotation reveals an unwanted use of non-canonical translation initiation codons. Mol Cell Proteomics 9:415–426PubMedCrossRefGoogle Scholar
  7. Cho BK, Charusanti P, Herrgard MJ, Palsson BO (2007) Microbial regulatory and metabolic networks. Curr Opin Biotechnol 18:360–364PubMedCrossRefGoogle Scholar
  8. Deanda K, Zhang M, Eddy C, Picataggio S (1996) Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl Environ Microbiol 62:4465–4470PubMedGoogle Scholar
  9. Devos D, Valencia A (2001) Intrinsic errors in genome annotation. Trends Genet 17:429–431PubMedCrossRefGoogle Scholar
  10. Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Applied Microbiol Biotechnol 63:258–266CrossRefGoogle Scholar
  11. Earl AM, Mohundro MM, Mian IS, Battista JR (2002) The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression. J Bacteriol 184:6216–6224PubMedCrossRefGoogle Scholar
  12. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM et al (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512PubMedCrossRefGoogle Scholar
  13. Gao GJ, Tian B, Liu LL, Sheng DH, Shen BH, Hua YJ (2003) Expression of Deinococcus radiodurans PprI enhances the radioresistance of Escherichia coli. DNA Repair 2:1419–1427PubMedCrossRefGoogle Scholar
  14. Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G (2006) Bio-ethanol–the fuel of tomorrow from the residues of today. Trends Biotechnol 24:549–556PubMedCrossRefGoogle Scholar
  15. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: Engineering plants and enzymes for biofuels production. Science 315:804–807PubMedCrossRefGoogle Scholar
  16. Jeffries TW (2005) Ethanol fermentation on the move. Nat Biotechnol 23(1):40–41PubMedCrossRefGoogle Scholar
  17. Jeon YJ, Svenson CJ, Joachimsthal EL, Rogers PL (2002) Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis. Biotechnol Lett 24:819–824CrossRefGoogle Scholar
  18. Joachimstahl E, Haggett KD, Jang JH, Rogers PL (1998) A mutant of Zymomonas mobilis ZM4 capable of ethanol production from glucose in the presence of high acetate concentrations. Biotechnol Lett 20:137–142CrossRefGoogle Scholar
  19. Kadar Z, Maltha SF, Szengyel Z, Reczey K, De Laat W (2007) Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH. Appl Biochem Biotechnol 137:847–858PubMedCrossRefGoogle Scholar
  20. Kerr AL, Jeon YJ, Svenson CJ, Rogers PL, Neilan BA (2010) DNA restriction-modification systems in the ethanologen, Zymomonas mobilis ZM4. Appl Microbiol Biotechnol 89:761–769PubMedCrossRefGoogle Scholar
  21. Kim IS, Barrow KD, Rogers PL (2000) Nuclear magnetic resonance studies of acetic acid inhibition of rec Zymomonas mobilis ZM4(pZB5). Appl Biochem Biotechnol 84–6:357–370CrossRefGoogle Scholar
  22. Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26PubMedCrossRefGoogle Scholar
  23. Kouvelis VN, Saunders E, Brettin TS, Bruce D, Detter C, Han C, Typas MA, Pappas KM (2009) Complete genome sequence of the ethanol producer Zymomonas mobilis NCIMB 11163. J Bacteriol 191:7140–7141PubMedCrossRefGoogle Scholar
  24. Lawford HG, Rousseau JD (1993) The effect of acetic acid on fuel ethanol-Production by Zymomonas. Appl Biochem Biotechnol 39:687–699CrossRefGoogle Scholar
  25. Lawford HG, Rousseau JD (1998) Improving fermentation performance of recombinant Zymomonas in acetic acid-containing media. Appl Biochem Biotechnol 70–2:161–172CrossRefGoogle Scholar
  26. Lawford HG, Rousseau JD, Tolan JS (2001) Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate. Appl Biochem Biotechnol 91–3:133–146CrossRefGoogle Scholar
  27. Lee K, Park J, Kim T, Yun H, Lee S (2010) The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies. Microb Cell Fact 9:94PubMedCrossRefGoogle Scholar
  28. Linger JG, Adney WS, Darzins A (2010) Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis. Appl Environ Microbiol 76:6360–6369PubMedCrossRefGoogle Scholar
  29. Liu Z, Blaschek H (2010) Biomass conversion inhibitors and in situ detoxification. In: Vertes A, Qureshi N, Yukawa H, Blaschek H (eds) Biomass to biofuels: strategies for global industries. Wiley, West Sussex, p 27Google Scholar
  30. Liu ZL, Moon J (2009) A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. Gene 446:1–10PubMedCrossRefGoogle Scholar
  31. Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW (2004) Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol 31:345–352PubMedGoogle Scholar
  32. Liu ZL, Slininger PJ, Gorsich SW (2005) Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl Biochem Biotechnol 121–124:451–460PubMedCrossRefGoogle Scholar
  33. Liu ZL, Moon J, Andersh BJ, Slininger PJ, Weber S (2008) Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. Applied Microbiol Biotechnol 81:743–753CrossRefGoogle Scholar
  34. Liu ZL, Ma M, Song M (2009) Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways. Mol Genet Genomics 282:233–244PubMedCrossRefGoogle Scholar
  35. MacLean D, Jones JD, Studholme DJ (2009) Application of ‘next-generation’ sequencing technologies to microbial genetics. Nat Rev 7:287–296Google Scholar
  36. McMillan JD (1994) Conversion of hemicellulose hydrolyzates to ethanol. In: Himmel ME, Baker JO, Overend RP (eds) Enzymatic conversion of biomass for fuels production, vol 566, ACS Symposium Series., pp 411–437CrossRefGoogle Scholar
  37. Metzker ML (2010) Sequencing technologies – the next generation. Nat Rev Genet 11:31–46PubMedCrossRefGoogle Scholar
  38. Mills T, Sandoval N, Gill R (2009) Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnol Biofuels 2:26PubMedCrossRefGoogle Scholar
  39. Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates II: inhibitors and mechanisms of inhibition. Biores Technol 74:25–33CrossRefGoogle Scholar
  40. Pan J, Wang J, Zhou ZF, Yan YL, Zhang W, Lu W, Ping S, Dai QL, Yuan ML, Feng B, Hou XG, Zhang Y, Ma R, Liu T, Feng L, Wang L, Chen M, Lin M (2009) IrrE, a global regulator of extreme radiation resistance in Deinococcus radiodurans, enhances salt tolerance in Escherichia coli and Brassica napus. PLoS One 4:2Google Scholar
  41. Panesar PS, Marwaha SS, Kennedy JF (2006) Zymomonas mobilis: an alternative ethanol producer. J Chem Technol Biotechnol 81:623–635CrossRefGoogle Scholar
  42. Parekh S, Vinci VA, Strobel RJ (2000) Improvement of microbial strains and fermentation processes. Appl Microbiol Biotechnol 54:287–301PubMedCrossRefGoogle Scholar
  43. Park JH, Lee SY, Kim TY, Kim HU (2008) Application of systems biology for bioprocess development. Trends Biotechnol 26:404–412PubMedCrossRefGoogle Scholar
  44. Patnaik R (2008) Engineering complex phenotypes in industrial strains. Biotechnol Prog 24:38–47PubMedCrossRefGoogle Scholar
  45. Payne SH, Huang ST, Pieper R (2010) A proteogenomic update to Yersinia: enhancing genome annotation. BMC Genomics 11:460PubMedGoogle Scholar
  46. Pienkos PT, Zhang M (2010) Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates. Cellulose 16:20Google Scholar
  47. Ranatunga TD, Jervis J, Helm RF, McMillan JD, Hatzis C (1997) Identification of inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation. Appl Biochem Biotechnol 67:185–198CrossRefGoogle Scholar
  48. Rogers PL, Jeon YJ, Lee KJ, Lawford HG (2007) Zymomonas mobilis for fuel ethanol and higher value products. In: Biofuels, vol 108. Advances in Biochemical Engineering/ Biotechnology. pp 263–288Google Scholar
  49. Seo JS, Chong HY, Park HS, Yoon KO, Jung C, Kim JJ, Hong JH, Kim H, Kim JH, Kil JI, Park CJ, Oh HM, Lee JS, Jin SJ, Um HW, Lee HJ, Oh SJ, Kim JY, Kang HL, Lee SY, Lee KJ, Kang HS (2005) The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol 23:63–68PubMedCrossRefGoogle Scholar
  50. Sittka A, Lucchini S, Papenfort K, Sharma CM, Rolle K, Binnewies TT, Hinton JC, Vogel J (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator. Hfq PLoS Genet 4:8CrossRefGoogle Scholar
  51. Stephanopoulos G (2007) Challenges in engineering microbes for biofuels production. Science 315:801–804PubMedCrossRefGoogle Scholar
  52. Takahashi CM, Takahashi DF, Carvalhal MLC, Alterthum F (1999) Effects of acetate on the growth and fermentation performance of Escherichia coli KO11. Appl Biochem Biotechnol 81:193–203PubMedCrossRefGoogle Scholar
  53. Tsui HC, Leung HC, Winkler ME (1994) Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol 13:35–49PubMedCrossRefGoogle Scholar
  54. Tyo KE, Alper HS, Stephanopoulos GN (2007) Expanding the metabolic engineering toolbox: more options to engineer cells. Trends Biotechnol 25:132–137PubMedCrossRefGoogle Scholar
  55. Valentin-Hansen P, Eriksen M, Udesen C (2004) The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 51:1525–1533PubMedCrossRefGoogle Scholar
  56. Viitanen PV, Tao L, Knoke K, Zhang Y, Caimi PG, Zhang M, Chou Y, Franden M (2009) Process for the production of ethanol from a medium comprising xylose, employing a recombinant Zymomonas strain having a reduced himA expression. Patent WO/2009/058938Google Scholar
  57. White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, Dodson RJ, Haft DH, Gwinn ML, Nelson WC, Richardson DL, Moffat KS, Qin H, Jiang L, Pamphile W, Crosby M, Shen M, Vamathevan JJ, Lam P, McDonald L, Utterback T, Zalewski C, Makarova KS, Aravind L, Daly MJ, Minton KW, Fleischmann RD, Ketchum KA, Nelson KE, Salzberg S, Smith HO, Venter JC, Fraser CM (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286:1571–1577PubMedCrossRefGoogle Scholar
  58. Widiastuti H, Kim JY, Selvarasu S, Karimi IA, Kim H, Seo JS, Lee DY (2010) Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis. Biotechnol Bioeng 108:655–665PubMedCrossRefGoogle Scholar
  59. Wright JC, Sugden D, Francis-McIntyre S, Riba-Garcia I, Gaskell SJ, Grigoriev IV, Baker SE, Beynon RJ, Hubbard SJ (2009) Exploiting proteomic data for genome annotation and gene model validation in Aspergillus niger. BMC Genomics 10:61PubMedCrossRefGoogle Scholar
  60. Yablonsky MD, Goodman AE, Stevnsborg N, Delima OG, Demorais JOF, Lawford HG, Rogers PL, Eveleigh DE (1988) Zymomonas mobilis CP4: a clarification of strains via plasmid profiles. J Biotechnol 9:71–79CrossRefGoogle Scholar
  61. Yang S, Pappas KM, Hauser LJ, Land ML, Chen G-L, Hurst GB, Pan C, Kouvelis V, Typas M, Pelletier DA, Klingeman DM, Chang Y-J, Samatova NF, Brown SD (2009a) Improved genome annotation for Zymomonas mobilis. Nat Biotechnol 27:893–894PubMedCrossRefGoogle Scholar
  62. Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M Jr, Brown SD (2009b) Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations. BMC Genomics 10:34PubMedCrossRefGoogle Scholar
  63. Yang S, Land ML, Klingeman DM, Pelletier DA, Lu T-YS, Martin SL, Guo HB, Smith JC, Brown SD (2010a) Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae. Proc Natl Acad Sci USA 107:10395–10400PubMedCrossRefGoogle Scholar
  64. Yang S, Pelletier DA, Lu TY, Brown SD (2010b) The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors. BMC Microbiol 10:135PubMedCrossRefGoogle Scholar
  65. Zhang M, Eddy C, Deanda K, Finkestein M, Picataggio S (1995) Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267:240–243PubMedCrossRefGoogle Scholar
  66. Zhang A, Wassarman KM, Rosenow C, Tjaden BC, Storz G, Gottesman S (2003) Global analysis of small RNA and mRNA targets of Hfq. Mol Microbiol 50:1111–1124PubMedCrossRefGoogle Scholar
  67. Zhang Y, Ma RQ, Zhao ZL, Zhou ZF, Lu W, Zhang W, Chen M (2010) irrE, an exogenous gene from Deinococcus radiodurans, improves the growth of and ethanol production by a Zymomonas mobilis strain under ethanol and acid stresses. J Microbiol Biotechnol 20:1156–1162PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  1. 1.Biosciences Division and BioEnergy Science Center, Oak Ridge National LaboratoryOak RidgeUSA

Personalised recommendations