Bioprocess and Biosystems Engineering

, Volume 31, Issue 1, pp 11–20 | Cite as

Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control

Original Paper


A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h−1, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.


Fed-batch technique Acetate formation High cell density Recombinant product formation Phosphotransferase system PTS mutations 



This work was sponsored by the Swedish Centre for Bioprocess Technology, CBioPT, which is gratefully acknowledged. We thank the Lundberg laboratory at the University of Gothenburg (Prof. Thomas Nyström and Dr Anne Farewell) and the University of Amsterdam (Profs. Peter Postma and Joost Teixeira de Mattos) for kindly supplying the strains and plasmids used in this work which were constructed in a joint EU Framework IV project: BIO4-CT98-0167.


  1. 1.
    Sandén AM, Boström M, Markland K, Larsson G (2005) Solubility and proteolysis of the Zb-MalE and Zb-MalE31 proteins during overproduction in Escherichia coli. Biotech Bioeng 90:239–247CrossRefGoogle Scholar
  2. 2.
    Boström M, Markland K, Sandén AM, Hedhammar M, Hober S, Larsson G (2005) Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli. Appl Microbiol Biotechnol 68:82–90CrossRefGoogle Scholar
  3. 3.
    Sandén AM, Prytz I, Tubulekas I, Förberg C, Le H, Hektor A, Neubauer P, Pragai Z, Harwood C, Ward A, Picon A, Teixeira de Mattos J, Postma P, Farewell A, Nyström T, Reeh S, Pedersen S, Larsson G (2003) Limiting factors in Escherichia coli fed-batch production of recombinant proteins. Biotech Bioeng 81:158–166CrossRefGoogle Scholar
  4. 4.
    Picon A, Teixeira de Mattos MJ, Postma PW (2005) Reducing the glucose uptake rate in Escherichia coli affects growth rate but not protein production. Biotech Bioeng 90:191–200CrossRefGoogle Scholar
  5. 5.
    Linn T, St.Pierre R (1990) Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J Bacteriol 172:1077–1084Google Scholar
  6. 6.
    Rose R (1998) The nucleotide sequence of pACYC184. Nucl Acid Res 16:355CrossRefGoogle Scholar
  7. 7.
    Larsson G, Törnkvist M (1996) Rapid sampling, cell inactivation and evaluation of low extracellular glucose concentrations during fed-batch cultivation. J Biotechnol 49:69–82CrossRefGoogle Scholar
  8. 8.
    Ferenci T (1996) Adaptation to lift at micromolar nutrient levels: the regulation of Eschrichia coli glucose transport by endoinduction and cAMP. FEMS Microbiol Rev 18:301–317CrossRefGoogle Scholar
  9. 9.
    Doelle H, Ewings K, Hollywood N (1982) Regulation of glucose metabolism in bacterial systems. Adv Biochem Eng 23:1–35Google Scholar
  10. 10.
    Meyer H-P, Leist C, Fiechter A (1984) Acetate formation in continuous culture of Escherichia coli K12 D1 on defined and complex media. J Biotechnol 1:355–358CrossRefGoogle Scholar
  11. 11.
    Gosset G (2005) Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system. Microb Cell Fact 4:14CrossRefGoogle Scholar
  12. 12.
    Curtis S, Epstein W (1975) Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. J Bacteriol 122:1189–1199Google Scholar
  13. 13.
    Duetz W, Rüedi L, Hermann R, O’Connor K, Büchs J, Witholt B (2000) Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl Env Micro 66:2641–2646CrossRefGoogle Scholar
  14. 14.
    John GT, Kliman I, Wittman C, Heinzle E (2003) Integrated optical sensing of dissolved oxygen in microtitre plates: a novel tool for microbial cultivation. Biotech Bioeng 81:829–836CrossRefGoogle Scholar
  15. 15.
    Jensen E, Carlsen S (1990) Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate and salts. Biotech Bioeng 36:1–11CrossRefGoogle Scholar
  16. 16.
    Chou C, Bennett G, San K (1994) Effect of modified glucose uptake using genetic engineering techniques on high-level recombinant protein in Escherichia coli dense cultures. Biotechnol Bioeng 44:952–960CrossRefGoogle Scholar
  17. 17.
    Kimata K, Inada T, Tagami H, Aiba H (1998) A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli. Mol Microbiol 29:1509–1519CrossRefGoogle Scholar
  18. 18.
    De Anda R, Lara A, Hernandez V, Hernandez-Montalvo V, Gosset G, Bolivar F, Ramirez O (2006) Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate. Metabolic Eng 8:281–290CrossRefGoogle Scholar
  19. 19.
    Cho S, Shin D, Ji GE, Heu S, Ryu S (2005) High-level recombinant protein production by overexpression of Mlc in Escherichia coli. J Biotechnol 119:197–203CrossRefGoogle Scholar
  20. 20.
    Boström M, Larsson G (2004) Process design for recombinant protein production based on the promoter, PmalK. Appl Microbiol Biotechnol 66:200–208CrossRefGoogle Scholar
  21. 21.
    Ryan W, Collier P, Loredo L, Pope J, Sachdev R (1996) Growth kinetics of Escherichia coli and expresiion of a recombinant protein and its isoforms under heat shock conditions. Biotechnol Prog 12:596–601CrossRefGoogle Scholar
  22. 22.
    Plumbridge J (1998) Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS. Mol Microbiol 27:369–380CrossRefGoogle Scholar
  23. 23.
    Plumbridge J (1999) Expression of the phosphotransferase system both mediates and is mediated by Mlc regulation in Escherichia coli. Mol Microbiol 33:260–273CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.School of BiotechnologyAlbaNova University Center, KTHStockholmSweden

Personalised recommendations