Microbial approach for the production of 5-hydroxytryptophan

Patent metadata

Field	Value
Publication number	US-10023889-B2
Application number	US-201515310040-A
Country	US
Kind code	B2
Filing date	May 14, 2015
Priority date	May 16, 2014
Publication date	Jul 17, 2018
Grant date	Jul 17, 2018

Abstract

5-hydroxytryptophan (5-HTP), a precursor of serotonin, is produced in a microbial host cell. A modified bacterial phenylalanine 4-hydroxylase (P4H) catalyzes the tryptophan 5-hydroxylation reaction. Optionally the host cell includes a cofactor regeneration mechanism, allowing continuous production of 5-HTP without supplementation of exogenous cofactors.

First claim

What is claimed is:

1. A genetically engineered bacterial cell comprising a modified bacterial phenylalanine-4-hydroxylase (P4H) that catalyzes the 5-hydroxylation of tryptophan in the presence of a tetrahydromonapterin (MH4) cofactor, wherein the modified P4H comprises a P4H from Xanthomonas campestris having an amino acid mutation at any one, any two, or all three of amino acid positions 98, 179, and 231 of SEQ ID NO: 1, or a modified P4H from Pseudomonas or Ralstonia , selected from the group consisting of: a P4H from P. aeruginosa comprising an amino acid mutation at any one, any two, or all three of amino acid positions 84, 162, and 213 of SEQ ID NO: 2, a P4H from P. putida having an amino acid mutation at any one, any two, or all three of amino acid positions 84, 162, and 213 of SEQ ID NO: 3, a P4H from P. fluorescence comprising an amino acid mutation at any one, any two, or all three of amino acid positions 84, 162, and 213 of SEQ ID NO: 4, or a P4H from R. eutropha H16 comprising an amino acid mutation at any one, any two, or all three of amino acid positions 113, 192, and 244 of SEQ ID NO: 5.

2. The genetically engineered bacterial cell of claim 1 which is genetically engineered to overproduce or accumulate tryptophan.

3. The genetically engineered bacterial cell of claim 1 further comprising a cofactor recycling system comprising at least one of a pterin-4a-carbinolamine dehydratase (PCD) and a dihydromonapterin reductase (DHMR).

4. The genetically engineered bacterial cell of claim 3 comprising a dihydromonapterin reductase encoded by the E. coli gene folM.

5. The genetically engineered bacterial cell of claim 1 wherein the modified bacterial P4H is derived from a Pseudomonas, Ralstonia , or Xanthomonas.

6. The genetically engineered bacterial cell of claim 1 wherein the modified P4H is selected from the group consisting of
(i) a P4H from X. campestris , comprising SEQ ID NO:1 modified by any one, any two, or all three amino acid mutations selected from the group consisting of L98Y, W179F and Y231C;
(ii) a P4H from P. aeruginosa comprising SEQ ID NO:2 modified by any one, any two, or all three amino acid mutations selected from the group consisting of L84Y, W162F, and Y213C;
(ii) a P4H from P. puticia comprising SEQ ID NO:3 modified by any one, any two, or all three amino acid mutations selected from the group consisting of L84Y, W162F, and Y213C;
(iv) a P4H from P. fluorescence comprising SEQ ID NO:4 modified by any one, any two, or all three amino acid mutations selected from the group consisting of L84Y, W162F, and Y213C; and
(v) a P4H from R. eutropha H16 comprising SEQ ID NO:5 modified by any one, any two, or all three amino acid mutations selected from the group consisting of L113Y, W192F, and Y244C.

7. The genetically engineered bacterial cell of claim 1 wherein the modified bacterial P4H further comprise at least one further mutation, said further mutation at any one, any two, or all three of amino acid positions 85, 223 and 282 of X. campestris P4H (SEQ ID NO: 1).

8. The genetically engineered bacterial cell of claim 1 comprising a first plasmid comprising a polynucleotide operably encoding (a) the modified bacterial P4H and (b) at least one of a pterin-4a-carbinolamine dehydratase (PCD) and a dihydromonapterin reductase (DHMR).

9. The genetically engineered bacterial cell of claim 8 further comprising a second plasmid comprising a polynucleotide operably encoding all or a portion of a trp operon.

10. The genetically engineered bacterial cell of claim 9 wherein the trp operon or portion thereof comprises a mutation S40F in TrpE.

11. The genetically engineered bacterial cell of claim 1 which is an Escherichia coli cell, a Bacillus subtilis cell, or a Corynebacterium glutamicum cell.

12. The genetically engineered bacterial cell of claim 1 which does not comprise a tetrahydrobiopterin (BH4) cofactor.

13. The genetically engineered bacterial cell of claim 1 which comprises an endogenous tetrahydrobiopterin (BH4) cofactor.

14. The genetically engineered bacterial cell of claim 1 which comprises an endogenous tetrahydromonapterin (MH4) cofactor.

15. A method of making 5-hydroxytryptophan (5-HTP) comprising:
culturing the genetically engineered bacterial cell of claim 1 and for a time sufficient to produce 5-HTP; and
isolating the 5-HTP.

16. The method of claim 15 further comprising purifying the 5-HTP.

17. The method of claim 15 further comprising incorporating the 5-HTP into a food product.

18. The method of claim 17 wherein the food product is an animal feed or beverage.

19. The method of claim 15 further comprising packaging the 5-HTP for sale.

20. The method of claim 19 further comprising providing instructions for use of the 5-HTP as a food additive, a food supplement, or a nutraceutical.

21. The method of claim 19 wherein the 5-HTP is packaged for use as an animal feed or beverage.

Assignees

• Univ Georgia

Inventors

• Yan Yajun
• Lin Yuheng

Classifications

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y105/01034

  6,7-Dihydropteridine reductase (1.5.1.34) · CPC title

• C12Y114/16001

  Phenylalanine 4-monooxygenase (1.14.16.1) · CPC title

• C12Y402/01096

  4a-Hydroxytetrahydrobiopterin dehydratase (4.2.1.96) · CPC title