Applied Microbiology and Biotechnology

, Volume 98, Issue 19, pp 8399–8411 | Cite as

Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production

  • Yongbo Yuan
  • Changhao Bi
  • Sergios A. Nicolaou
  • Kyle A. Zingaro
  • Matthew Ralston
  • Eleftherios T. PapoutsakisEmail author
Bioenergy and biofuels


A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5′ UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2’s ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.


Alcohol tolerance Lactobacillus plantarum Small noncoding RNA E. coli Overexpression Genomic integration 



This work was supported by a National Science Foundation Grant, CBET-1033926, and an Office of Naval Research (USA) Grant N000141010161.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2014_6004_MOESM1_ESM.pdf (58 kb)
ESM 1 (PDF 58 kb)


  1. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314:1565–1568. doi: 10.1126/science.1131969 PubMedCrossRefGoogle Scholar
  2. Block KF, Puerta-Fernandez E, Wallace JG, Breaker RR (2011) Association of OLE RNA with bacterial membranes via an RNA-protein interaction. Mol Microbiol 79:21–34. doi: 10.1111/j.1365-2958.2010.07439.x PubMedCrossRefPubMedCentralGoogle Scholar
  3. Borden JR, Jones SW, Indurthi D, Chen Y, Papoutsakis ET (2010) A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing. Metab Eng 12:268–281. doi: 10.1016/j.ymben.2009.12.004 PubMedCrossRefPubMedCentralGoogle Scholar
  4. Cho SH, Lei R, Henninger TD, Contreras LM (2014) Discovery of ethanol responsive small RNAs in Zymomonas mobilis. Appl Environ Microbiol. doi: 10.1128/aem.00429-14
  5. Couto JA, Pina C, Hogg T (1997) Enhancement of apparent resistance to ethanol in Lactobacillus hilgardii. Biotechnol Lett 19:487–490CrossRefGoogle Scholar
  6. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi: 10.1073/pnas.120163297 PubMedCrossRefPubMedCentralGoogle Scholar
  7. Du W, Brown JR, Sylvester DR, Huang J, Chalker AF, So CY, Holmes DJ, Payne DJ, Wallis NG (2000) Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria. J Bacteriol 182:4146–4152PubMedCrossRefPubMedCentralGoogle Scholar
  8. Dunlop MJ (2011) Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 4:32. doi: 10.1186/1754-6834-4-32 PubMedCrossRefPubMedCentralGoogle Scholar
  9. Fernandes P, Ferreira BS, Cabral JMS (2003) Solvent tolerance in bacteria: role of efflux pumps and cross-resistance with antibiotics. Int J Antimicrob Agents 22:211–216PubMedCrossRefGoogle Scholar
  10. Gaida SM, Al-Hinai MA, Indurthi DC, Nicolaou SA, Papoutsakis ET (2013) Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress. Nucleic Acids Res 41:8726–8737. doi: 10.1093/nar/gkt651 PubMedCrossRefPubMedCentralGoogle Scholar
  11. G-Alegria E, Lopez I, Ruiz JI, Saenz J, Fernandez E, Zarazaga M, Dizy M, Torres C, Ruiz-Larrea F (2004) High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol Lett 230:53–61PubMedCrossRefGoogle Scholar
  12. Gautam A, Rishi P, Tewari R (2011) UDP-N-acetylglucosamine enolpyruvyl transferase as a potential target for antibacterial chemotherapy: recent developments. Appl Microbiol Biotechnol 92:211–225. doi: 10.1007/s00253-011-3512-z PubMedCrossRefGoogle Scholar
  13. Gold RS, Meagher MM, Hutkins R, Conway T (1992) Ethanol tolerance and carbohydrate-metabolism in Lactobacilli. J Ind Microbiol 10:45–54CrossRefGoogle Scholar
  14. Goodarzi H, Bennett BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S (2010) Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli. Mol Syst Biol 6:378PubMedCrossRefPubMedCentralGoogle Scholar
  15. Gupta A, Singh R, Khare SK, Gupta MN (2006) A solvent tolerant isolate of Enterobacter aerogenes. Bioresour Technol 97:99–103PubMedCrossRefGoogle Scholar
  16. Hosokawa K, Park NH, Inaoka T, Itoh Y, Ochi K (2002) Streptomycin-resistant (rpsL) or rifampicin-resistant (rpoB) mutation in Pseudomonas putida KH146-2 confers enhanced tolerance to organic chemicals. Environ Microbiol 4:703–712Google Scholar
  17. Kameyama Y, Kawabe Y, Ito A, Kamihira M (2010) An accumulative site-specific gene integration system using cre recombinase-mediated cassette exchange. Biotechnol Bioeng 105:1106–1114. doi: 10.1002/bit.22619 PubMedGoogle Scholar
  18. Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MWEJ, Stiekema W, Lankhorst RMK, Bron PA, Hoffer SM, Groot MNN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Proc NatI Acad Sci U S A 100:1990–1995CrossRefGoogle Scholar
  19. Knoshaug EP, Zhang M (2009) Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 153:13–20PubMedCrossRefGoogle Scholar
  20. Marquardt JL, Siegele DA, Kolter R, Walsh CT (1992) Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase. J Bacteriol 174:5748–5752PubMedPubMedCentralGoogle Scholar
  21. Nicolaou SA, Gaida SM, Papoutsakis ET (2010) A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 12:307–331. doi: 10.1016/j.ymben.2010.03.004 PubMedCrossRefGoogle Scholar
  22. Nicolaou SA, Gaida SM, Papoutsakis ET (2011) Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes. Nucleic Acids Res 39:e152. doi: 10.1093/nar/gkr817 PubMedCrossRefPubMedCentralGoogle Scholar
  23. Nicolaou SA, Gaida SM, Papoutsakis ET (2012) Exploring the combinatorial genomic space in Escherichia coli for ethanol tolerance. Biotechnol J 7:1337–1345. doi: 10.1002/biot.201200227 PubMedCrossRefGoogle Scholar
  24. Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO (1991) Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Microbiol 57:893–900PubMedPubMedCentralGoogle Scholar
  25. Papoutsakis ET, Bi C, Nicolaou S (2011) Engineering complex microbial phenotypes with successive integrations of exogenous DNA (Siedna). USA Patent US2011300553-A1; WO2011153342-A2; WO2011153342-A3,Google Scholar
  26. Santos PM, Benndorf D, Sa-Correia I (2004) Insights into Pseudomonas putida KT2440 response to phenol-induced stress by quantitative proteomics. Proteomics 4:2640–2652PubMedCrossRefGoogle Scholar
  27. Venkataramanan KP, Jones SW, McCormick KP, Kunjeti SG, Ralston MT, Meyers BC, Papoutsakis ET (2013) The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum. BMC Genomics. 14 doi: 10.1186/1471-2164-14-849
  28. Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32:149–167. doi: 10.1111/j.1574-6976.2007.00094.x PubMedCrossRefGoogle Scholar
  29. Wilkins BM, Pritchard RH (1987) Escherichia coli and Salmonella typhimurium—cellular and molecular biology, Vol 1–2 - Neidhardt, Fc. Nature 330:707–708Google Scholar
  30. Woodruff LBA, Pandhal J, Ow SY, Karimpour-Fard A, Weiss SJ, Wright PC, Gill RT (2013) Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli. Metab Eng 15:124–133. doi: 10.1016/j.ymben.2012.10.007 PubMedCrossRefGoogle Scholar
  31. Zhou K, Zhou L, Lim Q, Zou R, Stephanopoulos G, Too HP (2011) Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol 12:18. doi: 10.1186/1471-2199-12-18 PubMedCrossRefPubMedCentralGoogle Scholar
  32. Zingaro KA, Papoutsakis ET (2012) Toward a semisynthetic stress response system to engineer microbial solvent tolerance. Mbio 3(5) doi: 10.1128/mBio.00308-12
  33. Zingaro KA, Papoutsakis ET (2013) 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 15:196–205. doi: 10.1016/j.ymben.2012.07.009
  34. Zingaro KA, Nicolaou SA, Papoutsakis ET (2013) Dissecting the assays to assess microbial tolerance to toxic chemicals in bioprocessing. Trends Biotechnol 31:643–653. doi: 10.1016/j.tibtech.2013.08.005 PubMedCrossRefGoogle Scholar
  35. Zingaro KA, Nicolaou SA, Yuan Y, Papoutsakis ET (2014) Exploring the heterologous genomic space for building, stepwise, complex, multicomponent tolerance to toxic chemicals. ACS Synth Biol 3:476–486. doi: 10.1021/sb400156v PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yongbo Yuan
    • 1
  • Changhao Bi
    • 1
    • 2
  • Sergios A. Nicolaou
    • 1
  • Kyle A. Zingaro
    • 1
  • Matthew Ralston
    • 1
  • Eleftherios T. Papoutsakis
    • 1
    Email author
  1. 1.Department of Chemical Engineering & Delaware Biotechnology InstituteUniversity of DelawareNewarkUSA
  2. 2.Tianjin Institute of BiotechnologyChinese Academy of SciencesTianjinChina

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