Publicação
Design and construction of a new biosynthetic pathway for the production of curcuminoids in Escherichia coli
| Resumo: | Curcuminoids are plant phenolic compounds with great anti-cancer potential. This thesis addresses the construction of an artificial biosynthetic pathway for curcuminoids production in Escherichia coli. This production was triggered by chemical or thermal induction to be used in industrial applications or bacterial therapies, respectively. To produce curcuminoids, including curcumin, from tyrosine, caffeic acid was produced as an intermediate in the pathway. The caffeic acid pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. TAL was able to efficiently convert tyrosine to p-coumaric acid and CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor; 280 mg/L of caffeic acid was produced. After validating the first steps of the pathway, curcuminoids production was studied. The best results were obtained with Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL1) and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1), yielding 70 mg/L of curcumin from ferulic acid. To produce curcumin through the caffeic acid pathway, caffeoyl-CoA O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.90 mg/L, 0.26 mg/L and 0.20 mg/L of curcumin were produced, respectively. Finally, caffeic acid and curcumin production was induced by heat using the dnaK heat shock promoter. Caffeic acid was successfully produced from tyrosine using TAL, C3H or CYP199A2 and the highest production was 14.41 mg/L. Regarding curcumin, 0.37 mg/L was produced from ferulic acid using 4CL1, DCS and CURS1. This is the first report on the in vivo use of DCS and CURS1 to produce curcuminoids. It was also demonstrated that curcumin can be produced from tyrosine using a pathway through the caffeic acid production. This alternative pathway represents a significant improvement in the heterologous production of curcuminoids using E. coli. In addition, caffeic acid and curcumin production in E. coli can be triggered by heat, thus suggesting its potential for the development of new bacterial therapies. |
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| Autores principais: | Rodrigues, Joana Lúcia Lima Correia |
| Assunto: | Engenharia e Tecnologia::Biotecnologia Industrial |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Curcuminoids are plant phenolic compounds with great anti-cancer potential. This thesis addresses the construction of an artificial biosynthetic pathway for curcuminoids production in Escherichia coli. This production was triggered by chemical or thermal induction to be used in industrial applications or bacterial therapies, respectively. To produce curcuminoids, including curcumin, from tyrosine, caffeic acid was produced as an intermediate in the pathway. The caffeic acid pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. TAL was able to efficiently convert tyrosine to p-coumaric acid and CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor; 280 mg/L of caffeic acid was produced. After validating the first steps of the pathway, curcuminoids production was studied. The best results were obtained with Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL1) and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1), yielding 70 mg/L of curcumin from ferulic acid. To produce curcumin through the caffeic acid pathway, caffeoyl-CoA O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.90 mg/L, 0.26 mg/L and 0.20 mg/L of curcumin were produced, respectively. Finally, caffeic acid and curcumin production was induced by heat using the dnaK heat shock promoter. Caffeic acid was successfully produced from tyrosine using TAL, C3H or CYP199A2 and the highest production was 14.41 mg/L. Regarding curcumin, 0.37 mg/L was produced from ferulic acid using 4CL1, DCS and CURS1. This is the first report on the in vivo use of DCS and CURS1 to produce curcuminoids. It was also demonstrated that curcumin can be produced from tyrosine using a pathway through the caffeic acid production. This alternative pathway represents a significant improvement in the heterologous production of curcuminoids using E. coli. In addition, caffeic acid and curcumin production in E. coli can be triggered by heat, thus suggesting its potential for the development of new bacterial therapies. |
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