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

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 Xow 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 qL-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.