title: Metabolic engineering of Escherichia coli for improving L-3,4-dihydroxyphenylalanine (L-DOPA) synthesis from glucose.
abstract: 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.
authors.affiliation: Departamento de Ingenierıa Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62210 Cuernavaca, Morelos, Mexico.
authors.forename: Ana Joyce
authors.initials: AJ
authors.lastname: Muñoz
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernández-Chávez
authors.forename: Ramon
authors.initials: R
authors.lastname: de Anda
authors.forename: Alfredo
authors.initials: A
authors.lastname: Martínez
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolívar
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gosset
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics / Q000378:metabolism; D002826:Chorismate Mutase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007980:Levodopa / Q000096:biosynthesis*; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011302:Prephenate Dehydratase / Q000235:genetics / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014174:Transketolase / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000096:biosynthesis; D017355:Zymomonas / Q000201:enzymology
chemical_list: D014443:Tyrosine; D007980:Levodopa; D006899:Mixed Function Oxygenases; D011303:Prephenate Dehydrogenase; C027017:cyclohexadienyl dehydrogenase; D014174:Transketolase; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase; D005947:Glucose