Synthesis of dopamine inE. coliusing plasmid-based expression system and its marked effect on host growth profiles

Abstract

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 Escherichia coli DH5α by the expression of 4-hydroxyphenylacetate-3-hydrolase (HpaBC) from E. coli 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 in vivo 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 in vivo 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.

Engineering Escherichia coli for High-Yielding Hydroxytyrosol Synthesis from Biobased l-Tyrosine

“…coli. Recently, HpaBC from E. coli has been reported to produce l -DOPA from l -tyrosine . Based on the report that coupling the native HpaBC from E.…”

“…coli with l -DOPA decarboxylase (DODC) from pig kidney cells achieved de novo synthesis of 27 mg/L dopamine from glucose in E. coli, we tried to introduce a bacterial DODC from P. putida in combination with HpaBC for dopamine production.…”

“…coli BL21 (DE3) harboring plasmids pET-DODC and pACYC-HpaBC, 4.5 mM (689.31 mg/L) dopamine with ∼90% conversion was obtained after 10 h (Figure S2A). According to a previous study, additional supplementation of iron and vitamin B6 could improve the activities of HpaBC from E.coli and DODC from pig kidney cells and result in enhanced dopamine production . Therefore, we also attempted to supplement iron and vitamin B6 to test whether dopamine could be further improved for the bacterial version of DODC.…”

“…Recently, HpaBC from E. coli has been reported to produce L-DOPA from L-tyrosine. 28 Based on the report that coupling the native HpaBC from E. coli with L-DOPA decarboxylase (DODC) from pig kidney cells achieved de novo synthesis of 27 mg/L dopamine from glucose in E. coli, 28 we tried to introduce a bacterial DODC from P. putida in combination with HpaBC for dopamine production. As shown in Figure S1, we investigated the enzyme cascade of HpaBC-DODC to biotransformation of L-tyrosine into dopamine.…”

“…According to a previous study, additional supplementation of iron and vitamin B6 could improve the activities of HpaBC from E.coli and DODC from pig kidney cells and result in enhanced dopamine production. 28 Therefore, we also attempted to supplement iron and vitamin B6 to test whether dopamine could be further improved for the bacterial version of DODC. As depicted in Figure S2B, addition of iron supplements and vitamin B6 did further boost the dopamine titer, reaching approximately 5 mM (773.56 mg/L) dopamine with >99% conversion.…”