Advertisement

Journal of Industrial Microbiology

, Volume 17, Issue 1, pp 47–52 | Cite as

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis inEscherichia coli

  • G Gosset
  • J Yong-Xiao
  • A Berry
Article

Abstract

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains ofEscherichia coli. In a strain having a wild-type PEP: glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA andpykF) resulted in a 3.4-fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of thetktA gene (encoding transketolase), thepykA and/orpykF mutations had no effect. A PTS glucose+ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS+ control strain. In the PTS glucose+ host background, overexpression oftktA caused a further 3.7-fold increase in carbon flow, while inactivation ofpykA andpykF caused a 5.8-fold increase. When all of the variables tested (PTS glucose+,pykA, pykF, and overexpressedtktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.

Keywords

amino acids aromatics E. coli DAHP PEP 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Beckwith J. 1987. The lactose operon. In:Escherichia coli andSalmonella typhimurium. Cellular and Molecular Biology, Vol 2 (Neidhardt FC, JL Ingraham, KB Low, B Magasanik, M Schaechter and HE Umbarger, eds), pp 1444–1452, American Society for Microbiology, Washington, DC.Google Scholar
  2. 2.
    Berry A, S Battist, G Chotani, T Dodge, S Peck, S Power and W Weyler. 1995. Biosynthesis of indigo using recombinantE. coli: development of a biological system for the cost-effective production of a large volume chemical. In: Proceedings of the Second Biomass Conference of the Americas: Energy, Environment, Agriculture, and Industry, pp 1121–1129, National Renewable Energy Laboratory. Golden, Colorado.Google Scholar
  3. 3.
    Bolivar F, RL Rodriguez, PJ Greene, MC Betlach, HL Heyneker and HW Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2: 95–113.PubMedGoogle Scholar
  4. 4.
    Chang ACY and SN Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134: 1141–1156.PubMedGoogle Scholar
  5. 5.
    Della-Cioppa G, SJ Garger, GG Sverlow, TH Turpen and LK Grill. 1990. Melanin production inEscherichia coli from a cloned tyrosinase gene. Bio/Technology 8: 634–638.PubMedGoogle Scholar
  6. 6.
    Draths KM, DL Pompliano, DL Conley, JW Frost, A Berry, GL Disbrow, RJ Staversky and JC Lievense. 1992. Biocatalytic synthesis of aromatics fromd-glucose: the role of transketolase. J Am Chem Soc 114: 3956–3962.Google Scholar
  7. 7.
    Draths KM, TL Ward and JW Frost. 1992. Biocatalysis and nineteenth century organic chemistry: conversion ofd-glucose into quinoid organics. J Am Chem Soc 114: 9725–9726.Google Scholar
  8. 8.
    Ensley BD, BJ Ratzkin, TD Osslund, MJ Simon, LP Wackett and DT Gibson. 1983. Expression of naphthalene oxidation genes inEscherichia coli results in biosynthesis of indigo. Science 222: 167–169.PubMedGoogle Scholar
  9. 9.
    Flores N, J Xiao, A Berry, F Bolivar and F Valle. 1996. Pathway engineering for the production of aromatic compounds inEscherichia coli. Nature Biotechnol 14: 620–623.Google Scholar
  10. 10.
    Frost JW and JC Lievense. 1994. Prospects for biocatalytic synthesis of aromatics in the 21 st century. New J Chem 18: 341–348.Google Scholar
  11. 11.
    Gubler M, M Jetten, SH Lee and AJ Sinskey. 1994. Cloning of the pyruvate kinase gene (pyk) ofCorynebacterium glutamicum and sitespecific inactivation ofpyk in a lysine-producingCorynebacterium lactofermentum strain. Appl Environ Microbiol 60: 2494–2500.PubMedGoogle Scholar
  12. 12.
    Lerner CG and M Inouye. 1990. Low copy number plasmids for regulated low-level expression of cloned genes inEscherichia coli with blue/white insert screening capability. Nucl Acids Res 18: 4631.PubMedGoogle Scholar
  13. 13.
    Mascarenhas D, DJ Ashworth and CS Chen. 1991. Deletion ofpgi alters tryptophan biosynthesis in a genetically engineered strain ofEscherichia coli. Appl Environ Microbiol 57: 2995–2999.PubMedGoogle Scholar
  14. 14.
    Miller JE, KC Backman, MJ O'Conner and RT Hatch. 1987. Production of phenylalanine and organic acids by phosphoenolpyruvate carboxylase-deficient mutants ofEscherichia coli. J Ind Microbiol 2: 143–149.Google Scholar
  15. 15.
    Miller JM. 1992. Preparation and use of P1vir lysates. In: A Short Course in Bacterial Genetics, pp 268–274, Cold Spring Harbor Laboratory, Cold Spring Harbor.Google Scholar
  16. 16.
    Patnaik R and JC Liao. 1994. Engineering ofEscherichia coli central metabolism for aromatic metabolite production with near theoretical yield. Appl Environ Microbiol 60: 3093–3098.Google Scholar
  17. 17.
    Patnaik R, WD Roof, RF Young and JC Liao. 1992. Stimulation of glucose catabolism inEscherichia coli by a potential futile cycle. J Bacteriol 174: 7527–7532.PubMedGoogle Scholar
  18. 18.
    Patnaik R, RG Spitzer and JC Liao. 1995. Pathway engineering for production of aromatics inExcherichia coli: confirmation of stoichiometric anlaysis by independent modulation of AroG, TktA, and Pps activities. Biotechnol Bioeng 46: 361–370.Google Scholar
  19. 19.
    Ponce E, N Flores, A Martinez, F Valle and F Bolivar. 1995. Cloning of the two pyruvate kinase isoenzyme structural genes fromEscherichia coli: the roles of these enzymes in pyruvate biosynthesis. J Bacteriol 177: 5719–5722.PubMedGoogle Scholar
  20. 20.
    Srinivasan PR and DB Sprinson. 1959. 2-keto-3-deoxy-d-arabo-heptonic acid 7-phosphate synthase. J Biol Chem 234: 716–722.PubMedGoogle Scholar
  21. 21.
    Wyman AR, LB Wolfe and D Botstein. 1985. Propagation of some human DNA sequences in bacteriophage λ vectors requires mutantE. coli hosts. Proc Natl Acad Sci 82: 2880–2884.PubMedGoogle Scholar
  22. 22.
    Yanisch-Perron C, J Vieira and J Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119.PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 1996

Authors and Affiliations

  • G Gosset
    • 1
  • J Yong-Xiao
    • 2
  • A Berry
    • 2
  1. 1.Instituto de BiotecnologiaUniversidad Nacional Autónoma de MéxicoCuernavacaMéxico
  2. 2.Genencor InternationalPalo AltoUSA

Personalised recommendations