Applied Microbiology and Biotechnology

, Volume 99, Issue 12, pp 5363–5371 | Cite as

Improving furfural tolerance of Zymomonas mobilis by rewiring a sigma factor RpoD protein

  • Fu-Rong Tan
  • Li-Chun Dai
  • Bo Wu
  • Han Qin
  • Zong-Xia Shui
  • Jing-Li Wang
  • Qi-Li Zhu
  • Qi-Chun Hu
  • Zhi-Yong Ruan
  • Ming-Xiong HeEmail author
Bioenergy and biofuels


Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ70, RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.


Furfural tolerance Ethanol production Global transcription machinery engineering Error-prone PCR Sigma factor RpoD protein 



This work was supported by Applied Basic Research Programs of Sichuan province (NO. 2014JY0065) and partially supported by Youth Science and Technology Foundation of Sichuan Province in China (Grant NO: 2015JQ0047). Open Funds of Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin (Tarim University, BRZD1403). Open Funds of State Key Laboratory of Microbial Technology (Shandong University, M2013-07), Open Funds of Key Laboratory of Microbial Resources Collection and Preservation (Ministry of Agriculture, MOA, 2013),


  1. Agrawal M, Chen RR (2011) Discovery and characterization of a xylose reductase from Zymomonas mobilis ZM4. Biotechnol Lett 33(11):2127–2133CrossRefPubMedGoogle Scholar
  2. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314(5805):1565–1568CrossRefPubMedGoogle Scholar
  3. Alper H, Stephanopoulos G (2007) Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab Eng 9(3):258–267CrossRefPubMedGoogle Scholar
  4. Barakat A, Monlau F, Steyer J-P, Carrere H (2012) Effect of lignin-derived and furan compounds found in lignocellulosic hydrolysates on biomethane production. Bioresour Technol 104:90–99CrossRefPubMedGoogle Scholar
  5. Barciszewski J, Siboska GE, Pedersen BO, Clark BF, Rattan SI (1997) A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA. FEBS Lett 414(2):457–460CrossRefPubMedGoogle Scholar
  6. Burgess RR, Anthony L (2001) How sigma docks to RNA polymerase and what sigma does. Curr Opin Microbiol 4(2):126–131CrossRefPubMedGoogle Scholar
  7. Chong HQ, Huang L, Yeow JW, Wang I, Zhang HF, Song H, Jiang RR (2013) Improving ethanol tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein(CRP). PLoS ONE 8(2):1–9CrossRefGoogle Scholar
  8. Conway T, Osman YA, Konnan JI, Hoffmann EM, Ingram LO (1987) Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J Bacteriol 169(3):949–954PubMedCentralPubMedGoogle Scholar
  9. Dunlop MJ (2011) Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 4(1):32–41CrossRefPubMedCentralPubMedGoogle Scholar
  10. Gardella T, Moyle H, Susskind MM (1989) A mutant Escherichia coli σ70 subunit of RNA polymerase with altered promoter specificity. J Mol Biol 206(4):579–590CrossRefPubMedGoogle Scholar
  11. Geddes CC, Peterson JJ, Mullinnix MT, Svoronos SA, Shanmugam KT, Ingram LO (2010a) Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse. Bioresour Technol 101(23):9128–9136CrossRefPubMedGoogle Scholar
  12. Geddes CC, Peterson JJ, Roslander C, Zacchi G, Mullinnix MT, Shanmugam KT, Ingram LO (2010b) Optimizing the saccharification of sugar cane bagasse using dilute phosphoric acid followed by fungal cellulases. Bioresour Technol 101(6):1851–1857CrossRefPubMedGoogle Scholar
  13. Geddes RD, Wang X, Yomano LP, Miller EN, Zheng H, Shanmugam KT, Ingram LO (2014) Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli Strain LY180. Appl Environ Microbiol 80(19):5955–5964CrossRefPubMedCentralPubMedGoogle Scholar
  14. Gorsich SW, Dien BS, Nichols NN, Slininger PJ, Liu ZL, Skory CD (2006) Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 71(3):339–349CrossRefPubMedGoogle Scholar
  15. He MX, Wu B, Shui ZX, Hu QC, Wang WG, Tan FR, Tang XY, Zhu QL, Pan K, Li Q (2012a) Transcriptome profiling of Zymomonas mobilis under furfural stress. Appl Microbiol Biotechnol 95(1):189–199CrossRefPubMedGoogle Scholar
  16. He MX, Wu B, Qin H, Ruan ZY, Tan FR, Wang JL, Shui ZX, Dai LC, Zhu QL, Pan K (2014) Zymomonas mobilis: a novel platform for future biorefineries. Biotechnol Biofuels 7(1):101–115CrossRefPubMedCentralPubMedGoogle Scholar
  17. He MX, Wu B, Shui ZX, Hu QC, Wang WG, Tan FR, Tang XY, Zhu QL, Pan K, Li Q, Su XH (2012b) Transcriptome profiling of Zymomonas mobilis under ethonal stress. Biotechnol Biofuels 5:75–84CrossRefPubMedCentralPubMedGoogle Scholar
  18. Hong SH, Lee J, Wood TK (2010a) Engineering global regulator Hha of Escherichia coli to control biofilm dispersal. Microb Biotechnol 3(6):717–728CrossRefPubMedCentralPubMedGoogle Scholar
  19. Hong SH, Wang X, Wood TK (2010b) Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H-NS of Escherichia coli. Microb Biotechnol 3(3):344–356CrossRefPubMedCentralPubMedGoogle Scholar
  20. Hristozova T, Gotcheva V, Tzvetkova B, Paskaleva D, Angelov A (2008) Effect of furfural on nitrogen assimilating enzymes of the lactose utilizing yeasts Candida blankii 35 and Candida pseudotropicalis 11. Enzym Microb Technol 43(3):284–288CrossRefGoogle Scholar
  21. Joachimsthal E, Rogers P (2000) Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures. In: Finkelstein M, Davison B (eds) Twenty-first symposium on biotechnology for fuels and chemicals. Applied Biochemistry and Biotechnology. Humana Press, pp 343–356Google Scholar
  22. Katahira S, Mizuike A, Fukuda H, Kondo A (2006) Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain. Appl Microbiol Biotechnol 72(6):1136–1143CrossRefPubMedGoogle Scholar
  23. Khan QA, Shamsi FA, Hadi S (1995) Mutagenicity of furfural in plasmid DNA. Cancer Lett 89(1):95–99CrossRefPubMedGoogle Scholar
  24. Klein-Marcuschamer D, Stephanopoulos G (2008) Assessing the potential of mutational strategies to elicit new phenotypes in industrial strains. Proc Natl Acad Sci U S A 105(7):2319–2324CrossRefPubMedCentralPubMedGoogle Scholar
  25. Lee JY, Sung BH, Yu BJ, Lee JH, Lee SH, Kim MS, Koob MD, Kim SC (2008) Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli. Nucleic Acids Res 36(16):102–111CrossRefGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4):402–408CrossRefPubMedGoogle Scholar
  27. 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(12):1867–1871CrossRefPubMedGoogle Scholar
  28. Kovach ME, Elzer PH, Steven Hill D, Robertson GT, Farris MA, II Martin Roop R, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefPubMedGoogle Scholar
  29. Mackenzie KF, Eddy CK, Ingram LO (1989) Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc. J Bacteriol 171(2):1063–1068PubMedCentralPubMedGoogle Scholar
  30. Miller EN, Jarboe LR, Turner PC, Pharkya P, Yomano LP, York SW, Nunn D, Shanmugam K, Ingram LO (2009) Furfural inhibits growth by limiting sulfur assimilation in ethanologenic Escherichia coli strain LY180. Appl Environ Microbiol 75(19):6132–6141CrossRefPubMedCentralPubMedGoogle Scholar
  31. NCBI conserved domain (2015) Search for conserved domains within a protein or coding nucleotide sequence.Google Scholar
  32. Neale AD, Scopes RK, Kelly JM, Wettenhall REH (1986) The two alcohol dehydrogenases of Zymomonas mobilis purification by differential dye ligand chromatography, molecular characterisation and physiological roles. Eur J Biochem 154:119–124CrossRefPubMedGoogle Scholar
  33. Nichols NN, Hector RE, Saha BC, Frazer SE, Kennedy GJ (2014) Biological abatement of inhibitors in rice hull hydrolyzate and fermentation to ethanol using conventional and engineered microbes. Biomass Bioenergy 67:79–88CrossRefGoogle Scholar
  34. Owens JT, Miyake R, Murakami K, Chmura AJ, Fujita N, Ishihama A, Meares CF (1998) Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. Proc Natl Acad Sci U S A 95(11):6021–6026CrossRefPubMedCentralPubMedGoogle Scholar
  35. Parawira W, Tekere M (2011) Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review. Crit Rev Biotechnol 31(1):20–31CrossRefPubMedGoogle Scholar
  36. Park KS, Lee DK, Lee H, Lee Y, Jang YS, Kim YH, Yang HY, Lee SI, Seol W, Kim JS (2003) Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat Biotechnol 21(10):1208–1214CrossRefPubMedGoogle Scholar
  37. Rogers PL, Jeon YJ, Lee KJ, Lawford HG (2007) Zymomonas mobilis for fuel ethanol and higher value products. Adv Biochem Eng Biotechnol 108:263–288PubMedGoogle Scholar
  38. Sharma UK, Ravishankar S, Shandil RK, Praveen P, Balganesh T (1999) Study of the interaction between bacteriophage T4 asiA and Escherichia coli ς70, using the yeast two-hybrid system: neutralization of asiA toxicity to E. coli cells by coexpression of a truncated ς70 fragment. J Bacteriol 181(18):5855–5859PubMedCentralPubMedGoogle Scholar
  39. Shi DJ, Wang CL, Wang KM (2009) Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 36(1):139–147CrossRefPubMedGoogle Scholar
  40. Sridhar M, Kiran Sree N, Venkateswar Rao L (2002) Effect of UV radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains. Bioresour Technol 83(3):199–202CrossRefPubMedGoogle Scholar
  41. Tao F, Miao JY, Shi GY, Zhang KC (2005) Ethanol fermentation by an acid-tolerant Zymomonas mobilis under non-sterilized condition. Process Biochem 40(1):183–187CrossRefGoogle Scholar
  42. Tokiwa Y, Calabia BP (2008) Biological production of functional chemicals from renewable resources. Can J Chem 86(6):548–555CrossRefGoogle Scholar
  43. Uppugundla N, da Costa Sousa L, Chundawat SPS, Yu X, Simmons B, Singh S, Gao X, Kumar R, Wyman CE, Dale BE, Balan V (2014) A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover. Biotechnol Biofuels 7:72–86CrossRefPubMedCentralPubMedGoogle Scholar
  44. Wang X, Yomano LP, Lee JY, York SW, Zheng H, Mullinnix MT, Shanmugam K, Ingram LO (2013) Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proc Natl Acad Sci U S A 110(10):4021–4026CrossRefPubMedCentralPubMedGoogle Scholar
  45. Yang S, Pan C, Tschaplinski TJ, Hurst GB, Engle NL, Zhou W, Dam P, Xu Y, Rodriguez M Jr, Dice L, Johnson CM, Davison BH, Brown SD (2013) Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses. PLoS ONE 8(7):68886–68899CrossRefGoogle Scholar
  46. Zhang Y, Ma R, Zhao Z, Zhou Z, 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(7):1156–1162CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Fu-Rong Tan
    • 1
  • Li-Chun Dai
    • 1
  • Bo Wu
    • 1
  • Han Qin
    • 1
  • Zong-Xia Shui
    • 1
  • Jing-Li Wang
    • 1
  • Qi-Li Zhu
    • 1
  • Qi-Chun Hu
    • 1
    • 2
  • Zhi-Yong Ruan
    • 3
  • Ming-Xiong He
    • 1
    • 2
    Email author
  1. 1.Biogas Institute of Ministry of AgricultureBiomass Energy Technology Research CentreChengduPeople’s Republic of China
  2. 2.Key Laboratory of Development and Application of Rural Renewable EnergyMinistry of AgricultureChengduPeople’s Republic of China
  3. 3.Institute of Agricultural Resources and Regional PlanningChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

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