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Engineering Escherichia coli for efficient cellobiose utilization

Abstract

Escherichia coli normally cannot utilize the β-glucoside sugar cellobiose as a carbon and energy source unless a stringent selection pressure for survival is present. The cellobiose-utilization phenotype can be conferred by mutations in the two cryptic operons, chb and asc. In this study, the cellobiose-utilization phenotype was conferred to E. coli by replacing the cryptic promoters of these endogenous operons with a constitutive promoter. Evolutionary adaptation of the engineered strain CP12CHBASC by repeated subculture in cellobiose-containing minimal medium led to an increase in the rate of cellobiose uptake and cell growth on cellobiose. An efficient cellobiose-metabolizing E. coli strain would be of great importance over glucose-metabolizing E. coli for a simultaneous saccharification and fermentation process, as the cost of the process would be reduced by eliminating one of the three enzymes needed to hydrolyze cellulose into simple sugars.

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References

  1. Adin DM, Visick KL, Stabb EV (2008) Identification of a cellobiose utilization gene cluster with cryptic beta-galactosidase activity in Vibrio fischeri. Appl Environ Microbiol 74:4059–4069. doi:https://doi.org/10.1128/aem.00190-08

  2. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Engin 10:305–311. doi:https://doi.org/10.1016/j.ymben.2007.08.003

  3. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462. doi:https://doi.org/10.1126/science.277.5331.1453

  4. Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of FLP-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14. doi:https://doi.org/10.1016/0378-1119(95)00193-a

  5. Clomburg JM, Gonzalez R (2010) Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology. Appl Microbiol Biotechnol 86:419–434. doi:https://doi.org/10.1007/s00253-010-2446-1

  6. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645

  7. Ghim CM, Kim T, Mitchell RJ, Lee SK (2010) Synthetic biology for biofuels: building designer microbes from the scratch. Biotechnol Bioproce Eng 15:11–21. doi:https://doi.org/10.1007/s12257-009-3065-5

  8. Ha S-J, Galazka JM, Rin Kim S, Choi J-H, Yang X, Seo J-H, Louise Glass N, Cate JHD, Jin Y-S (2011) Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc Natl Acad Sci USA 108:504–509. doi:https://doi.org/10.1073/pnas.1010456108

  9. Hall BG (1999) Transposable elements as activators of cryptic genes in E. coli. Genetica 107:181–187

  10. Hall BG, Betts PW (1987) Cryptic genes for cellobiose utilization in natural isolates of Escherichia coli. Genetics 115:431–439

  11. Hall BG, Xu L (1992) Nucleotide- sequence, function, activation and evolution of the cryptic asc operon of Escherichia coli K- 12. Mol Biol Evol 9:688–706

  12. Hall BG, Betts PW, Kricker M (1986) Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state. Mol Biol Evol 3:389–402

  13. Jarboe LR, Zhang XL, Wang X, Moore JC, Shanmugam KT, Ingram LO (2010) Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J Biomed Biotechnol. doi: 76104210.1155/2010/761042

  14. Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64:82–87

  15. Kachroo AH, Kancherla AK, Singh NS, Varshney U, Mahadevan S (2007) Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. Mol Microbiol 66:1382–1395. doi:https://doi.org/10.1111/j.1365-2958.2007.05999.x

  16. La Grange D, den Haan R, van Zyl W (2010) Engineering cellulolytic ability into bioprocessing organisms. Appl Microbiol Biotechnol 87:1195–1208. doi:https://doi.org/10.1007/s00253-010-2660-x

  17. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19:556–563. doi:https://doi.org/10.1016/j.copbio.2008.10.014

  18. Lee SM, Jin LH, Kim JH, Han SO, Na HB, Hyeon T, Koo YM, Kim J, Lee JH (2010) Beta-glucosidase coating on polymer nanofibers for improved cellulosic ethanol production. Bioprocess Biosyst Eng 33:141–147. doi:https://doi.org/10.1007/s00449-009-0386-x

  19. Lynd LR, van Zyl WH, McBride JE, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16:577–583. doi:https://doi.org/10.1016/j.copbio.2005.08.009

  20. Moniruzzaman M, Lai X, York S, Ingram L (1997) Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon. App Environ Microbiol 63:4633–4637

  21. Mukerji M, Mahadevan S (1997) Cryptic genes: evolutionary puzzles. J Genet 76:147–159

  22. Negrete A, Ng W-I, Shiloach J (2010) Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21). Microb Cell Fact 9:75

  23. Panesar PS, Marwaha SS, Kennedy JF (2006) Zymomonas mobilis: an alternative ethanol producer. J Chem Technol Biotechnol 81:623–635. doi:https://doi.org/10.1002/jctb.1448

  24. Peralta-Yahya PP, Keasling JD (2010) Advanced biofuel production in microbes. Biotechnol J 5:147–162. doi:https://doi.org/10.1002/biot.200900220

  25. Plumbridge J, Pellegrini O (2004) Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP. Mol Microbiol 52:437–449. doi:https://doi.org/10.1111/j.1365-2958.2004.03986.x

  26. Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, del Cardayre SB, Keasling JD (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559–562, http://www.nature.com/nature/journal/v463/n7280/suppinfo/nature08721_S1.html

  27. Stephanopoulos G (2007) Challenges in engineering microbes for biofuels production. Science 315:801–804. doi:https://doi.org/10.1126/science.1139612

  28. van Rooyen R, Hahn-Hägerdal B, La Grange DC, van Zyl WH (2005) Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains. J Biotechnol 120:284–295. doi:https://doi.org/10.1016/j.jbiotec.2005.06.013

  29. Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E (2010) Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol 87:1303–1315. doi:https://doi.org/10.1007/s00253-010-2707-z

  30. Wen F, Nair NU, Zhao H (2009) Protein engineering in designing tailored enzymes and microorganisms for biofuels production. Curr Opin Biotechnol 20:412–419

  31. Xu Q, Singh A, Himmel ME (2009) Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose. Curr Opin Biotechnol 20:364–371. doi:https://doi.org/10.1016/j.copbio.2009.05.006

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Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Education, Science, and Technology (MEST) of the Korean Government (NRF-2009-C1AAA001-2009-0093479); by the Basic Science Research Program, through the NRF, funded by the MEST (NRF-2009-0076912); and by the World Class University (WCU) program, through the Korea Science and Engineering Foundation, funded by the MEST (R31-2008-000-20012-0).

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Correspondence to Sung Kuk Lee.

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Vinuselvi, P., Lee, S.K. Engineering Escherichia coli for efficient cellobiose utilization. Appl Microbiol Biotechnol 92, 125–132 (2011). https://doi.org/10.1007/s00253-011-3434-9

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Keywords

  • chb operon
  • Cellobiose metabolism
  • asc operon
  • Cryptic genes
  • Escherichia coli