Host cells and methods for producing hydroxytyrosol

Patent metadata

Field	Value
Publication number	US-11932896-B2
Application number	US-201816232614-A
Country	US
Kind code	B2
Filing date	Dec 26, 2018
Priority date	Jun 24, 2016
Publication date	Mar 19, 2024
Grant date	Mar 19, 2024

Abstract

The present invention provides for a composition comprising: (a) a first host cell capable of producing L-DOPA; and (b) a modified host cell is capable of converting L-DOPA into hydroxytyrosol (HTy); wherein any one or both of the first host cell and second host cell is a genetically modified host cell.

First claim

We claim:

1. A cell co-culture composition comprising a first Escherichia coli strain that produces levopdopa (L-DOPA); and a second Escherichia coli strain that converts L-DOPA produced by the first Escherichia coli strain into hydroxytyrosol (HTy), wherein the first Escherichia coli strain comprises a set of expression constructs to express the genes set forth in (a) not shared with the second strain and the second Escherichia coli strain comprises a set of expression constructs to express the genes set forth in (b) not shared with the first strain, wherein:
(a) the first Escherichia coli strain expresses endogenous FolE, FoIX, and FoIM genes and comprises: a first nucleic acid construct that expresses a mouse tyrosine hydroxylase (TH) comprising the amino acid sequence set forth in SEQ NO:1, or a variant thereof having TH activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; a second nucleic acid construct that expresses a human dihydropteridine reductase (DHPR) comprising the amino acid sequence set forth in SEQ NO:3, or a variant thereof having DHPR activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; and
a third nucleic acid that expresses a human pterin-4-alpha-carbinolamine dehydratase (PCD) comprising the amino acid sequence set forth in SEQ NO:2, or a variant thereof having PCD activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and wherein each of the first, second, and third nucleic acid is operably linked to a promoter; and the first Escherichia coli strain further expresses the following enzymes for the synthesis of L-tyrosine:
(i) a feedback resistant mutant D146N of a DAHP synthase (AroG) having AroG activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and a feedback resistant mutant M53I A354V of a chorismate mutase/prephenate dehydrogenase (TryA) having TyrA activity and at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; wherein a nucleic acid construct encoding the feedback resistant mutant D146N of the DAHP synthase and a nucleic acid construct encoding the feedback resistance mutant M53I A254V of the chorismate mutase/prephenate dehydrogenase are each operably linked to a promoter and integrated at the pykF locus; and
(ii) phosphoenolpyruvate synthase (PpsA), transketolase A (TktA), DHQ synthase (AroB), DHQ dehydratase (AroD), quinate/shikimate dehydrogenase (YdiB), shikimate dehydrogenase (AroE), shikimate kinase I/II (AroK/L), EPSP synthase (AroA), chorismate synthase (AroC), and tyrosine aminotransferase (TyrB); and

(b) the second Escherichia coli host cell strain is a strain in which the endogenous feaB gene is deleted, and comprises: a first nucleic acid construct that expresses a pig L-DOPA decarboxylase (DDC) comprising the amino acid sequence set forth in SEQ NO:5, or a variant thereof having DDC activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; and a second nucleic acid construct that expresses a Micrococcus luteus monoamine oxidase (MAO) comprising the amino acid sequence set forth in SEQ ID NO:6, or a variant thereof having MAO activity that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; wherein each of the first and second nucleic acid is operably linked to a promoter.

2. The cell co-culture composition of claim 1 , wherein the first Escherichia coli strain produces 2.0 mM or more of L-tyrosine when grown or cultured in a M9Y defined medium (1% glucose).

3. The cell co-culture composition of claim 2 , wherein the first Escherichia coli strain produces 2.6 mM or more of L-tyrosine when-grown or cultured in a M9Y defined medium (1% glucose).

4. A method for producing hydroxytyrosol (HTy) comprising:
culturing the cell co-culture composition of claim 1 , wherein HTy is produced;
optionally extracting or separating the HTy from the co-culture.

5. The cell co-culture composition of claim 1 , wherein the TH comprises the amino acid sequence set forth in SEQ ID NO:1; the DHPR comprises the amino acid sequence set forth in SEQ ID NO:3; the PCD comprises the amino acid sequence set forth in SEQ ID NO:2; the DDC comprises the amino acid sequence set forth in SEQ ID NO:5; and the MAO comprises the amino acid sequence set forth in SEQ ID NO:6.

6. The cell co-culture composition of claim 1 , wherein the feedback resistant mutant D146N of the DAHP synthase comprises the amino acid sequence set forth in SEQ ID NO:7 and the feedback resistant mutant M531 A354V of the chorismate mutase/prephenate dehydrogenase comprises the amino acid sequence set forth in SEQ ID NO:8.

Assignees

• Univ California

Inventors

• Yoshida Erika
• Lee Taek Soon

Classifications

• C12P7/22 Primary

  aromatic · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12P13/22

  Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine · CPC title

• C12N2510/02

  Cells for production · CPC title

• C12P39/00

  Processes involving microorganisms of different genera in the same process, simultaneously · CPC title