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