Heterogeneous expression of DOPA decarboxylase to improve the production of dopamine in Escherichia coli / 生物工程学报

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.

Inhibition activities of levodopa derivatives against dopa decarboxylase / 中国药学杂志

OBJECTIVE: To investigate the inhibition activities of levodopa(LD) derivatives against dopa decarboxylase(DDC). METHODS: Five compounds 1-4a/4b were synthesized and identified as 1,1-dimethyl-3-carboxyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (1), 1α-methyl-3-carboxyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline(2),3-carboxyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline(3), 1α/1α-phenyl-3-carboxyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (4a/4b). Their inhibition activities against DDC were assayed, and Michael's equation of 1 were further tested. RESULTS: Compounds 1-4 could inhibit DDC activities dose dependency, while 1 showed strongest activity with its 50% inhibition concentration to be 0.387 mmol·L-1. Compound 1 showed non-competitive inhibition model with LD against DDC enzyme. CONCLUSION: Compounds 1-4 are proved to inhibit DDC activities for the first time. Therefore, compounds 1-3 of Mucuna seeds could improve LD bioavailability effectively in part through this mechanism.

Study on relationship between single nucleotide polymorphisms polymorphisms of DDC and DRD1 and clinical phenotype feature in autistic children / 中国医师杂志

Objective To investigate the association between single nucleotide polymorphisms (SNPs) of DOPA decarboxylase (DDC) and dopamine receptor-1 (DRD1) and clinical phenotype feature in autistic children.Methods TaqMan probes real-time polymerase chain reaction (PCR) was used to determine genotype and allele of SNPs of DDC gene (rs6592961) and DRD1 (rs251937) gene in 97 autism children.The Children Autism Rating Scale (CARS) was used to evaluate clinical phenotype feature.Results There was no significant difference in the distribution of the allelic frequency and genotype between mild-medium group and severe group of CARS scores (P ＞ 0.05).For DDC gene (rs6592961),significant difference was found in subscale between genotypes G/G and A/A (P =0.043).For DRD1 gene (rs251937),significant difference was found in subscale between genotypes T/T and C/C (P =0.029).Conclusions In DDC gene (rs6592961),the children with G/G genotype were more obvious than the children with A/A genotype.In DRD1 gene (rs251937),the children with T/T genotype were more obvious than the children with C/C genotype.

In Vitro Release of L-dopa and Dopamine by Genetically Modified Fibroblasts of the Rat

A construct DNA containing the cDNA for rat tyrosine hydroxylase(TH) and another construct DNA containing the cDNA for bovine L-dopa decarboxylase(DDC) were made and those genes were transferred by calcium phosphate transfection method into the immortalized rat fibroblasts. A both TH- & DDC-positive clone was identified by immunocytochemical staining. These cells produced L-dopa and dopamine and released them into the cell culture medium in vitro, which was confirmed by high performance liquid chromatography. These results have general implications for the application of gene therapy and specific implications for Parkinson disease.

In Vitro Release of L-dopa and Dopamine by Genetically Modified Fibroblasts of the Rat

A construct DNA containing the cDNA for rat tyrosine hydroxylase(TH) and another construct DNA containing the cDNA for bovine L-dopa decarboxylase(DDC) were made and those genes were transferred by calcium phosphate transfection method into the immortalized rat fibroblasts. A both TH- & DDC-positive clone was identified by immunocytochemical staining. These cells produced L-dopa and dopamine and released them into the cell culture medium in vitro, which was confirmed by high performance liquid chromatography. These results have general implications for the application of gene therapy and specific implications for Parkinson disease.