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- Nachgewiesen in: USPTO Patent Applications
- Sprachen: English
- Document Number: 20240132921
- Publication Date: April 25, 2024
- Appl. No: 18/263894
- Application Filed: February 08, 2022
- Assignees: SILICOLIFE LDA. (Braga, PT)
- Claim: 1. A method for production of tyrosol, wherein a transgenic bacterial cell that heterologously expresses: a. phenylpyruvate decarboxylase (ARO10) and that overexpresses each of: b. phospho-2-dehydro-3-deoxyheptonate aldolase (aroF) c. prephenate dehydrogenase (tyrA) and wherein each of the following genes is not expressed: i. pheAL (bifunctional chorismate mutase/prephenate dehydratase) ii. feaB (phenylacetaldehyde dehydrogenase) is grown in a medium comprising a metabolic precursor of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), particularly wherein the metabolic precursor is glucose, and optionally, phenylalanine as a supplement; and tyrosol is extracted from said medium.
- Claim: 2. The method according to claim 1, wherein the transgenic bacterial cell is of the genus Escherichia, particularly wherein the transgenic bacterial cell is of the species E. coli, more particularly wherein the transgenic bacterial cell is of the strain E. coli BL21.
- Claim: 3. The method according to claim 1, wherein the gene encoding the phenylpyruvate decarboxylase originates from yeast, particularly from S. cerevisiae.
- Claim: 4. A method for production of salidroside, wherein a transgenic bacterial cell according to claim 1 additionally heterologously expresses uridine diphosphate dependent glycosyltransferase (UGT85A1), and the cell is grown in a medium comprising a metabolic precursor of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), particularly glucose, and optionally, phenylalanine as a supplement; and salidroside is extracted from said medium.
- Claim: 5. The method according to claim 4, wherein the gene encoding uridine diphosphate dependent glycosyltransferase originates from a plant, particularly from Arabidopsis, more particularly from A. thaliana.
- Claim: 6. The method according to claim 1, wherein the transgenic bacterial cell does not overexpress any of the following proteins: alcohol dehydrogenase, DNA-binding transcriptional regulatory protein (tyrR), and tyrosine aminotransferase.
- Claim: 7. The method according to claim 1, wherein the only heterologously expressed genes of the transgenic bacterial cell are i) wherein the method is directed at the production of tyrosol, the only heterologously expressed gene in the cell is phenylpyruvate decarboxylase; ii) wherein the method is directed at the production of salidroside, the only heterologously expressed genes in the cell are phenylpyruvate decarboxylase and uridine diphosphate dependent glycosyltransferase.
- Claim: 8. The method according to claim 1, wherein the overexpressed genes and the transgenes are introduced into the transgenic bacterial cell via one or several plasmid vector(s), particularly wherein phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase and prephenate dehydrogenase are encoded by a medium-copy plasmid vector, and/or uridine diphosphate dependent glycosyltransferase is encoded by a low-copy plasmid vector.
- Claim: 9. The method according to claim 1, wherein said transgenic bacterial cell comprises one or more plasmids encoding said heterologously expressed or overexpressed enzymes under control of a promoter sequence operable in said cell, particularly a T7 promoter (SEQ ID NO. 31), a lac promoter (SEQ ID NO. 32), a tac promoter (SEQ ID NO. 33) or a trc promoter (SEQ ID NO. 34), more particularly wherein the gene encoding uridine diphosphate dependent glycosyltransferase is under control of a trc promoter, and/or the gene encoding phenylpyruvate decarboxylase is under control of a T7 promoter, and/or the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase is under control of a T7 promoter, and/or the gene encoding prephenate dehydrogenase is under control of a T7 promoter.
- Claim: 10. The method according to claim 9, wherein the expression of said heterologous and/or overexpressed genes is induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG), particularly at a concentration of ˜0.1 mM IPTG for 96 h.
- Claim: 11. The method according to claim 1, wherein said medium comprises 10 to 50 g/L of glucose, particularly 15 to 30 g/L of glucose.
- Claim: 12. The method according to claim 1, wherein the transgenes are codon-optimized for expression in said transgenic bacterial cell.
- Claim: 13. The method according to claim 1, wherein the medium comprises: 5-10 g/L Na2HPO4·2H2O, 2-4 g/L KH2PO4, 0.25-1 g/L NaCl, 0.5-1.5 g/L NH4Cl, 1-3% (w/v) glucose, 0.01-0.05% (w/v) yeast extract, 3-7 mM MgSO4, 0.005-0.02 g/L CaCl2 and antibiotics, particularly wherein the antibiotics are 50-200 μg/mL ampicillin, 10-50 μg/mL kanamycin and 25-45 μg/mL chloramphenicol.
- Claim: 14. The method according to claim 1, wherein a. the phenylpyruvate decarboxylase has at least 60%, 65%, 70%, 75%, 80%, particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% sequence identity with SEQ ID NO 1, and wherein the phenylpyruvate decarboxylase has a catalytic activity of at least 75% of the activity of SEQ ID NO 1 and/or b. the phospho-2-dehydro-3-deoxyheptonate aldolase has at least 60%, 65%, 70%, 75%, 80%, particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% sequence identity with SEQ ID NO 2 and wherein the phospho-2-dehydro-3-deoxyheptonate aldolase has a catalytic activity of at least 75% of the activity of SEQ ID NO 2 and/or c. the prephenate dehydrogenase has at least 60%, 65%, 70%, 75%, 80%, particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% sequence identity with SEQ ID NO 3 and wherein the prephenate dehydrogenase has a catalytic activity of at least 75% of the activity of SEQ ID NO 3 and/or d. the uridine diphosphate dependent glycosyltransferase has at least 60%, 65%, 70%, 75%, particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% sequence identity with SEQ ID NO 4 and wherein the uridine diphosphate dependent glycosyltransferase has a catalytic activity of at least 75% of the activity of SEQ ID NO 4.
- Claim: 15. A transgenic cell according to claim 1.
- Current International Class: 12; 12; 12; 12; 12; 12
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