RESUMEN
The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca(2+)-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca(2+)-dependent conformational change. The Ca(2+)-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca(2+)-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca(2+) conditions. Because TyrA activity exhibits robust changes in response to Ca(2+)-dependent-EF-hand conformational alterations, TyrA could be a good Ca(2+)-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca(2+)-dependent conformational change.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Calcio/metabolismo , Secuencia Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Prefenato Deshidrogenasa/química , Prefenato Deshidrogenasa/genética , Prefenato Deshidrogenasa/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.