Publicação
Exploiting synthetic biology for the production of prenylflavonoids in Escherichia coli
| Resumo: | Prenylflavonoids are flavonoids bearing a prenylated sidechain. Among them, xanthohumol and prenylnaringenin (PN) isomers, including 3-prenylnaringenin (3-PN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) have attracted interest due to their bioactive properties. However, their low accumulation in plants limits their utilization. The construction of microbial cell factories able to produce them appeared as an alternative solution. Herein, we intended to engineer Escherichia coli strains able to produce PN compounds and xanthohumol using glucose as main substrate. Firstly, a step-by-step optimization was performed to construct an efficient pathway for de novo production of the precursors naringenin chalcone and naringenin. E. coli M-PAR-121 expressing naringenin chalcone pathway composed by tyrosine ammonia lyase (TAL) from Flavobacterium johnsoniae (FjTAL), 4-coumarate:CoA ligase 1 (4CL-1) from Arabidopsis thaliana (At4CL), and chalcone synthase (CHS) from Curcubita maxima (CmCHS) was able to produce 560.2 mg/L of naringenin chalcone. E. coli M-PAR-121 expressing naringenin biosynthetic pathway composed by FjTAL, At4CL, CmCHS, and chalcone isomerase (CHI) from Medicago sativa (MsCHI) was able to produce 769.5 mg/L of naringenin [1]. These production levels were the highest reported in E. coli until this moment. Since the final steps of the prenylflavonoids pathway depend on the availability of dimethylallyl pyrophosphate (DMAPP) and/or S-adenosylmethionine (SAM), clustered regularly interspaced short palindromic repeats (CRISPR) methodologies were exploited to improve the intracellular availability of both compounds. E. coli M-PAR-121 strain with the integration of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from E. coli (EcDXS) (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic prenyltransferase (PT) from Streptomyces roseochromogenes (CloQ) (pCDFDuet_CloQ) and pRSFDuet_FjTAL_CmCHS_At4CL_MsCHI was selected as the best PN producer strain, being able to produce 135.33 mg/L of 3-PN and 8.72 mg/L of 6-PN [2]. Moreover, E. coli M-PAR-121:BlIDI:metK, constructed by integrating SAM synthase (metK) and isopentenyl diphosphate isomerase (IDI) from Bacillus licheniformis (BlIDI) was selected as the best xanthohumol producing strain. Xanthohumol production reached 5.26 mg/L when pRSFDuet_FjTAL_CmCHS_At4CL was expressed in combination with CdpC3PT from Neosartorya fischeri and O-methyltransferase from Humulus lupulus (HlOMT1) (pCDFDuet_CdpC3PT_HlOMT1). These productions represent the highest reported to date |
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| Autores principais: | Gomes, Daniela Filipa Correia |
| Outros Autores: | Rodrigues, Joana Lúcia Lima Correia; Scrutton, Nigel S.; Rodrigues, L. R. |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | outro |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Prenylflavonoids are flavonoids bearing a prenylated sidechain. Among them, xanthohumol and prenylnaringenin (PN) isomers, including 3-prenylnaringenin (3-PN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) have attracted interest due to their bioactive properties. However, their low accumulation in plants limits their utilization. The construction of microbial cell factories able to produce them appeared as an alternative solution. Herein, we intended to engineer Escherichia coli strains able to produce PN compounds and xanthohumol using glucose as main substrate. Firstly, a step-by-step optimization was performed to construct an efficient pathway for de novo production of the precursors naringenin chalcone and naringenin. E. coli M-PAR-121 expressing naringenin chalcone pathway composed by tyrosine ammonia lyase (TAL) from Flavobacterium johnsoniae (FjTAL), 4-coumarate:CoA ligase 1 (4CL-1) from Arabidopsis thaliana (At4CL), and chalcone synthase (CHS) from Curcubita maxima (CmCHS) was able to produce 560.2 mg/L of naringenin chalcone. E. coli M-PAR-121 expressing naringenin biosynthetic pathway composed by FjTAL, At4CL, CmCHS, and chalcone isomerase (CHI) from Medicago sativa (MsCHI) was able to produce 769.5 mg/L of naringenin [1]. These production levels were the highest reported in E. coli until this moment. Since the final steps of the prenylflavonoids pathway depend on the availability of dimethylallyl pyrophosphate (DMAPP) and/or S-adenosylmethionine (SAM), clustered regularly interspaced short palindromic repeats (CRISPR) methodologies were exploited to improve the intracellular availability of both compounds. E. coli M-PAR-121 strain with the integration of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from E. coli (EcDXS) (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic prenyltransferase (PT) from Streptomyces roseochromogenes (CloQ) (pCDFDuet_CloQ) and pRSFDuet_FjTAL_CmCHS_At4CL_MsCHI was selected as the best PN producer strain, being able to produce 135.33 mg/L of 3-PN and 8.72 mg/L of 6-PN [2]. Moreover, E. coli M-PAR-121:BlIDI:metK, constructed by integrating SAM synthase (metK) and isopentenyl diphosphate isomerase (IDI) from Bacillus licheniformis (BlIDI) was selected as the best xanthohumol producing strain. Xanthohumol production reached 5.26 mg/L when pRSFDuet_FjTAL_CmCHS_At4CL was expressed in combination with CdpC3PT from Neosartorya fischeri and O-methyltransferase from Humulus lupulus (HlOMT1) (pCDFDuet_CdpC3PT_HlOMT1). These productions represent the highest reported to date |
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