Homeostatic regulation of l-dopa biosynthesis

Patent metadata

Field	Value
Publication number	US-2019314313-A1
Application number	US-201716341222-A
Country	US
Kind code	A1
Filing date	Oct 11, 2017
Priority date	Oct 11, 2016
Publication date	Oct 17, 2019
Grant date	—

Abstract

Disclosed herein are methods and compositions for the production of L-3,4-dihydroxyphenylalanine from a bacteria.

First claim

1 . A genetically engineered cell capable of producing L-3,4-dihydroxyphenylalanine (L-DOPA), wherein said cell comprises a gene encoding PP2551 of Pseudomonas putida.

2 . The cell of claim 1 , wherein an amino acid sequence encoded by PP2251 of Pseudomonas putida comprises SEQ ID NO: 1.

3 . The cell of claim 1 , further comprising a promoter recognized by PP2251.

4 . The cell of claim 3 , wherein the promoter recognized by PP2251 comprises SEQ ID NO: 2.

5 . The cell of claim 1 , wherein the cell further comprises genes hpaB and hpaC encoding HpaB and HpaC respectively.

6 . The cell of claim 5 , wherein the amino acid sequence encoded by hpaB is SEQ ID NO: 3.

7 . The cell of claim 5 , wherein the amino acid sequence encoded by hpaC is SEQ ID NO: 4.

8 . The cell of claim 1 , wherein the cell is capable of producing L-DOPA at a steady state.

9 . The cell of claim 1 , wherein transcriptional regulator tyrosine repressor (tyrR) has been deleted.

10 . The cell of claim 1 , wherein transcriptional regulator carbon storage regulator A (csrA) has been deleted.

11 . The cell of claim 1 , wherein glucose transport system of the bacterium has been altered from phosphotransferase system (PTS) to ATP-dependent uptake.

12 . The cell of claim 1 , wherein phosphorylation system of the cell has been altered to overexpress galactose permease gene (galP) and glucokinase gene (glk).

13 . The cell of claim 1 , wherein glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) have been knocked out.

14 . The cell of claim 1 , wherein a fusion protein chimera of a downstream pathway of chorismate has been integrated.

15 . A plasmid comprising a gene encoding PP2551 of Pseudomonas putida , a promoter thereof, and genes encoding hpaB and hpaC.

16 . A cell line comprising the plasmid of claim 16 .

17 . A method of producing L-DOPA, comprising transforming a cell with a gene encoding PP2551 of Pseudomonas putida.

18 . The method of claim 17 , wherein an amino acid sequence encoded by PP2251 of Pseudomonas putida comprises SEQ ID NO: 1.

19 . The method of claim 17 , further comprising a promoter recognized by PP2251.

20 . The method of claim 19 , wherein the promoter recognized by PP2251 comprises SEQ ID NO: 2.

21 . (canceled)

22 . (canceled)

23 . (canceled)

Assignees

• Univ Texas

Inventors

• Ellington Andrew D
• THYER Ross

Classifications

• C12N2800/101

  for bacteria · CPC title

• C12N2330/51

  Specially adapted vectors · CPC title

• C12N15/78

  for Pseudomonas · CPC title

• C12P13/06

  Alanine; Leucine; Isoleucine; Serine; Homoserine · CPC title

• A61K31/198 Primary

  Alpha-amino acids, e.g. alanine or edetic acid [EDTA] (betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00) · CPC title