Metabolic engineering of Escherichia coli for improving l-3,4-dihydroxyphenylalanine (l-DOPA) synthesis from glucose

  • Ana Joyce Muñoz
  • Georgina Hernández-Chávez
  • Ramon de Anda
  • Alfredo Martínez
  • Francisco Bolívar
  • Guillermo GossetEmail author
Original Paper


l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS gluc+ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q l-Tyr in the PTS+ and the PTS gluc+ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS gluc+ tyrR strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS gluc+ tyrR phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.


E. coli Aromatics l-tyrosine l-DOPA Phosphotransferase system TyrR transcriptional dual regulator 



This work was supported by CONACyT grants 83039 and 126793. AJM was supported by a fellowship from CONACyT. We thank Luz María Martínez, Mercedes Enzaldo, Juan Manuel Hurtado, Mario Trejo and Martín Patiño for technical assistance.


  1. 1.
    Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315PubMedCrossRefGoogle Scholar
  2. 2.
    Báez JL, Bolívar F, Gosset G (2001) Determination of 3-deoxy-d-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system. Biotechnol Bioeng 73:530–535PubMedCrossRefGoogle Scholar
  3. 3.
    Balderas-Hernández VE, Sabido-Ramos A, Silva P, Cabrera-Valladares N, Hernández-Chávez G, Báez-Viveros JL, Martínez A, Bolívar F, Gosset G (2009) Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli. Microb Cell Fact 8:1–12CrossRefGoogle Scholar
  4. 4.
    Chavez-Bejar MI, Lara AR, Lopez H, Hernandez-Chavez G, Martinez A, Ramirez OT, Bolivar F, Gosset G (2008) Metabolic engineering of Escherichia coli for l-tyrosine production by the expression of genes coding for the chorismate mutase domain of the native chorismate mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from Zymomonas mobilis. Appl Environ Microbiol 74:3284–3290PubMedCrossRefGoogle Scholar
  5. 5.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645PubMedCrossRefGoogle Scholar
  6. 6.
    De Anda R, Lara AR, Hernandez V, Hernandez-Montalvo V, Gosset G, Bolıvar F, Ramırez OT (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. Metab Eng 8:281–290PubMedCrossRefGoogle Scholar
  7. 7.
    Flores N, Yong-Xiao J, Berry A, Bolívar F, Valle F (1996) Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Biotechnol 14:620–623PubMedCrossRefGoogle Scholar
  8. 8.
    Flores S, Gosset G, Flores N, de Graff AA, Bolívar F (2002) Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by 13C labeling and NMR spectroscopy. Metab Eng 4:124–137PubMedCrossRefGoogle Scholar
  9. 9.
    Förberg C, Eliaeson T, Häggström L (1988) Correlation of theoretical and experimental yields of phenylalanine from non-growing cells of a rec Escherichia coli strain. J Biotechnol 7:319–332CrossRefGoogle Scholar
  10. 10.
    Gosset G, Yong-Xiao J, Berry A (1996) A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli. J Ind Microbiol 17:47–52PubMedCrossRefGoogle Scholar
  11. 11.
    Hernández-Montalvo V, Martínez A, Hernández-Chávez G, Bolivar F, Valle F, Gosset G (2003) Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products. Biotechnol Bioeng 83:687–694PubMedCrossRefGoogle Scholar
  12. 12.
    Ho PY, Chıou MS, Chao AC (2003) Production of l-DOPA by tyrosinase immobilized on modified polystyrene. Appl Biochem Biotechnol 111:139–152PubMedCrossRefGoogle Scholar
  13. 13.
    Koyanagi T, Katayama T, Suzuki H, Nakazawa H, Yokozeki K, Kumagai H (2005) Effective production of 3, 4-dihydroxyphenyl l-alanine (l-dopa) with Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR. J Biotechnol 115:303–306PubMedCrossRefGoogle Scholar
  14. 14.
    Kramer M, Kremer-Muschen S, Wubbolts MG (2006) Process for the preparation of l-3, 4-dihydroxyphenylalanine by aerobic fermentation of a microorganism. US Patent 2006/0141587A1Google Scholar
  15. 15.
    Krishnaveni R, Rathod V, Thakur MS, Neelgund YF (2009) Transformation of l-tyrosine to L-dopa by a novel fungus, Acremonium rutilum, under submerged fermentation. Curr Microbiol 58:122–128PubMedCrossRefGoogle Scholar
  16. 16.
    Lee JY, Xun L (1998) Novel biological process for L-DOPA production from l-tyrosine by p-hydroxyphenylacetate 3-hydroxylase. Biotechnol Lett 20:479–482CrossRefGoogle Scholar
  17. 17.
    Lee SG, Hong SP, Sung MH (1999) Development of an enzymatic system for the production of dopamine from catechol, pyruvate and ammonia. Enzyme Microb Technol 25:268–302Google Scholar
  18. 18.
    Lütke-Eversloh T, Stephanopoulos G (2007) l-Tyrosine production by deregulated strains of Escherichia coli. Appl Microbiol Biotechnol 75:103–110PubMedCrossRefGoogle Scholar
  19. 19.
    Lütke-Eversloh T, Stephanopoulos G (2008) Combinatorial pathway analysis for improved l-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression. Metab Eng 10:69–77PubMedCrossRefGoogle Scholar
  20. 20.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring, NY, p 68Google Scholar
  21. 21.
    Patnaik R, Liao JC (1994) Engineering of Escherichia coli central metabolism for aromatic production with near theoretical yield. Appl Environ Microbiol 60:3903–3908PubMedGoogle Scholar
  22. 22.
    Patnaik R, Spitzer RG, Liao JC (1995) Pathway engineering for production of aromatics in Escherichia coli: confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities. Biotechnol Bioeng 46:361–370PubMedCrossRefGoogle Scholar
  23. 23.
    Pittard J, Camakaris H, Yang J (2005) The TyrR regulon. Mol Microbiol 55:16–26PubMedCrossRefGoogle Scholar
  24. 24.
    Prieto MA, Garcıa JL (1994) Molecular characterization of 4-hydroxyphenylacetate 3-hydroxylase of Escherichia coli. A two-protein component enzyme. J Biol Chem 269:22823–22829PubMedGoogle Scholar
  25. 25.
    Qi WW, Vannelli T, Breinig S, Ben-Bassat A, Gatenby AA, Haynie SL, Sariaslani FS (2007) Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene. Metab Eng 9:268–276PubMedCrossRefGoogle Scholar
  26. 26.
    Reinhold DF, Utne T, Abramson NL (1987) Process for L-dopa. US patent 4716246Google Scholar
  27. 27.
    Snell KD, Draths KM, Frost JW (1996) Synthetic modification of the Escherichia coli chromosome: enhancing the biocatalytic conversion of glucose into aromatic chemicals. J Am Chem Soc 118:5605–5614CrossRefGoogle Scholar
  28. 28.
    Takai A, Nishi R, Joe Y, Ito H (2005) l-Tyrosine producing bacterium and a method for producing l-tyrosine. US Patent application no. 2005/0277179A1Google Scholar

Copyright information

© Society for Industrial Microbiology 2011

Authors and Affiliations

  • Ana Joyce Muñoz
    • 1
  • Georgina Hernández-Chávez
    • 1
  • Ramon de Anda
    • 1
  • Alfredo Martínez
    • 1
  • Francisco Bolívar
    • 1
  • Guillermo Gosset
    • 1
    Email author
  1. 1.Departamento de Ingenierıa Celular y BiocatálisisInstituto de Biotecnología, Universidad Nacional Autónoma de MéxicoCuernavaca, MorelosMexico

Personalised recommendations