Patent ID: 11932896
Assignee: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.