Applied Microbiology and Biotechnology

, Volume 42, Issue 1, pp 100–107 | Cite as

Characterization and evaluation of a pta (phosphotransacetylase) negative mutant of Escherichia coli HB101 as production host of foreign lipase

  • D. H. Hahm
  • J. Pan
  • J. S. Rhee
Applied Genetics and Regulation Original paper


In order to evaluate the pta(phosphotransacetylase) (−) mutant of Escherichia coli as a potential host of foreign lipase expression, the pta(−) mutant HB101 was constructed for the purpose of blocking the acetate synthetic pathway. Since acetate is known as a major inhibitory by-product of cell growth and foreign protein production, the growth characteristics and expression kinetics of the microbial lipase of the pta(−) E. coli mutant were investigated. The growth rate was considerably decreased (about 30%) when grown on M9 minimal media containing glucose, mannose or glycerol. Growth retardation was not observed when a gluconeogenic carbon source (acetate, malate or succinate) was utilized. It should be noted that the growth rate of the mutant was enhanced (about 20%) in modified M9 media including a gluconeogenic carbon source and NZ-amine. Growth inhibition of the pta(−) mutant by menadione, a representative redox-cycling drug, was more pronounced than that of the parental type of E. coli. Furthermore, the inhibition effect was more pronounced in glucose minimal medium, whereas the menadione sensitivity was not observed when a gluconeogenic carbon source was used as a sole carbon source or the lactate dehydrogenase gene from Lactobacillus casei was introduced in the pta(−) mutant. Therefore, it is suggested that the growth deficiency of the pta(−) mutant is closely related to the intracellular redox balance. When the pseudomonad lipase was expressed in the pta(−) mutant, a comparable expression rate and yield to the parental type strain was observed. High-cell-density culture if the mutant was easy to achieve even under the fluctuating conditions of residual glucose concentration.


Lipase Lactobacillus Minimal Medium Menadione Lactobacillus Casei 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andersen KB, Meyenburg KV (1980) Are growth rates of E. coli in batch cultures limited by respiration? J Bacteriol 144:114–123Google Scholar
  2. Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymatic interconversion of acetate and acetyl CoA in E. coli. J Gen Microbiol 102:327–336Google Scholar
  3. Brown SW, Meyer H-P, Fiechter A (1985) Continuous production of human leukocyte interferon with E. coli and continuous cell lysis in two stage chemostat. Appl Microbiol Biotechnol 23:5–9Google Scholar
  4. Bauer KA, Ben-Bassat A, Dawson M, De La Puente VT, Deway JO (1990) Improved expression of human interleukin-2 in high-cell-density fermentor culture cultures of E. coli K-12 by a phosphotransacetylase mutant. Appl Environ Microbiol 56:1296–1302PubMedGoogle Scholar
  5. Chung GH, Lee YP, Yoo OJ, Rhee JS (1991a) Overexpression of thermostable lipase gene from Pseudomonas fluorescens SIK W1 in E.coli. Appl Microbiol Biotechnol 35:237–241Google Scholar
  6. Chung GH, Lee YP, Jeohn GH, Yoo OJ, Rhee JS (1991b) Cloning and nucleotide sequence of a thermostable lipase gene from Pseudomonas fluorescens SIK W1. Agric Biol Chem 55:2359–2365Google Scholar
  7. Dannelly HK, Roseman S (1992) NAD+ and NADH regulate an ATP-dependent kinase that phosphorylates enzyme I of the E.coli phosphotransferase system. Proc Natl Acad Sci USA 89:11274–11276Google Scholar
  8. Dedhia N, Hottiger T, Chen W, Bailey JE (1992) Genetic manipulation of central carbon metabolism in E.coli. In: Ladisch MR, Bose A (eds) Proceedings of the 9th International Biotechnology Symposium, August 16–21, Crystal City, Virginia. American Chemical Society, Washington, DC, pp 59–62Google Scholar
  9. Diaz-Ricci JC, Regan L, Bailey JE (1991) Effect of alteration of acetic acid synthesis pathway on the fermentation pattern of E.coli. Biotechnol Bioeng 38:1318–1324Google Scholar
  10. El-Mansi EMT, Holms WH (1989) Control of carbon flux to acetate excretion during growth of E.coli in batch and continuous cultures J Gen Microbiol 135:2875–2883PubMedGoogle Scholar
  11. Falk M, Korz D, Rantze E, Schultze R, Sanders E (1989) Fermentation with recombinant micro-organisms. Sci Annu Rep 67–69Google Scholar
  12. Greenberg JT, Demple B (1989) A global response induced in E. coli by redox cycling agents overlaps with that induced by peroxide stress. J Bacteriol 171:3933–3939Google Scholar
  13. Han K, Lim HC, Hong J (1992) Acetic acid formation in E. coli fermentation. Biotechnol Bioeng 39:663–671Google Scholar
  14. Hanahan D (1985) DNA Cloning, vol I. IRL Press, OxfordGoogle Scholar
  15. Holms WH (1986) The central metabolic pathways of E. coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Curr Top Cell Regul 28:69–104Google Scholar
  16. Kim SF, Baek SJ, Pack MY (1991) Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene. Appl Environ Microbiol 57:2413–2417Google Scholar
  17. Konstantinov KB, Nishio N, Yoshida T (1990) Glucose feeding strategy accounting for the decreasing oxidative capacity of recombinant E.coli in fed-batch cultivation for phenylalanine production. J Ferment Bioeng 70:253–260Google Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  19. Landwall P, Holme T (1977) Influence of glucose and dissolved oxygen concentrations on yields of E. coli B in dialysis culture. J Gen Microbiol 103:353–358Google Scholar
  20. Luli GW, Strohl WR (1990) Comparison of growth, acetate production, and acetate inhibition of E. coli strains in batch and fed-batch fermentations. Appl Environ Microbiol 56:1004–1011PubMedGoogle Scholar
  21. Majewski RA, Domach MM (1990) Simple constrained-optimization view of acetate overflow in E. coli. Biotechnol Bioeng 35:732–738Google Scholar
  22. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  23. McCleary WR, Stock JB, Ninfa AJ (1993) Is acetyl phosphate a global signal in E. coli? J Bacteriol 175:2793–2798Google Scholar
  24. Neidhardt FC, Ingraham JL, Schaechter M (1990) Physiology of the bacterial cells, Chapter 5. Sinauer Associates, Mass., USAGoogle Scholar
  25. Pan JG, Rhee JS, Lebeault JM (1987) Physiological constraints in increasing biomass concentration of E. coli B in fed-batch culture. Biotechnol Lett 9:89–94Google Scholar
  26. Riesenberg D (1991) High-cell-density cultivation of E. coli Curr Biol 2:380–384Google Scholar
  27. Riesenberg D, Schulz V, Knorre WA, Pohl H-D, Korz D, Sanders EA, Ross A, Deckwer W-D (1991) High-cell-density cultivation of E.coli at controlled specific growth rate. J Biotechnol 20:17–28Google Scholar
  28. Rinas U, Kracke-Helm HA, Schügerl K (1989) Glucose as a substrate in recombinant strain fermentation technology. Appl Microbiol Biotechnol 31:163–167Google Scholar
  29. Wanner BL, Wilmes-Riesenberg MR (1992) Involvement of phosphotracetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in E.coli. J Bacteriol 174:2124–2130Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • D. H. Hahm
    • 1
  • J. Pan
    • 2
  • J. S. Rhee
    • 1
  1. 1.Department of BiotechnologyKorea Advanced Institute of Science and Technology (KAIST)TaejonKorea
  2. 2.Bioprocess Research Group, Genetic Engineering Research InstituteKorea Institute of Science and TechnologyTaejonKorea

Personalised recommendations