Synthesis of dopamine in <i>E. coli</i> using plasmid-based expression system and its marked effect on host growth profiles

<p>L-Dopa and dopamine are important pathway intermediates toward the synthesis of catecholamine such as epinephrine and norepinephrine from amino acid L-tyrosine. Dopamine, secreted from dopaminergic nerve cells, serves as an important neurotransmitter. We report the synthesis of dopamine by extending the aromatic amino acid pathway of <i>Escherichia coli</i> DH5α by the expression of 4-hydroxyphenylacetate-3-hydrolase (HpaBC) from <i>E. coli</i> and an engineered dopa decarboxylase (DDC) from pig kidney cell. The activity of HpaBC and DDC require 200 µM iron supplementation and 50 µM vitamin B6, respectively as additives to the growth media. The maximum concentration of L-dopa and dopamine obtained from the broth was around 26 and 27 mg/L after 24 hr of separate shake flask studies. We observed that in the presence of dopamine synthesized <i>in vivo</i> host growth was remarkably enhanced. These observations lead us to an interesting finding about the role of these catecholamines on bacterial growth. It is clear that synthesis of dopamine <i>in vivo</i> actually promotes growth much efficiently as compared to when dopamine is added to the system from outside. From HPLC and GC–MS data it was further observed that L-dopa was stable within the observable time of experiments whereas dopamine actually was subjected to degradation via oxidation and host consumption.</p

Metabolic engineering of <i>Escherichia coli</i> W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol

<p>L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in <i>Escherichia coli</i> is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.</p