Optimized microbial cells for production of melatonin and other compounds

Patent metadata

Field	Value
Publication number	US-10851365-B2
Application number	US-201716089977-A
Country	US
Kind code	B2
Filing date	Mar 30, 2017
Priority date	Mar 31, 2016
Publication date	Dec 1, 2020
Grant date	Dec 1, 2020

Abstract

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.

First claim

The invention claimed is:

1. A variant of E. coli GTP cyclohydrolase I (GCH1) having at least 94% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16 and comprising one or more mutations, wherein,
in an E. coli cell comprising a pterin-4α-carbinolamine dehydratase (PCD) and at least one of a tryptophan hydroxylase (TPH), a tyrosine hydroxylase (TH) and a phenylalanine hydroxylase (PheH), the variant provides for an increased hydroxylation activity of at least one of the TPH, TH and PheH as compared to native E. coli GCH1,
wherein at least one of the one or more mutations is in an amino acid residue selected from the group consisting of D97, M99, T101, V102, A125, K129, N170, V179, T196, T198, S199, L200, S207, H212, E213, F214, L215 and H221, and
wherein the mutation in N170 is N170K, N170D or N170L; the mutation in T198 is T198I, T198V or T198L; the mutation in V179 is V179A; the mutation in H212 is H212R or H212K; and the mutation in H221 is H221R or H221K.

2. The variant of claim 1 , wherein the variant
comprises a mutation selected from D97V, D97L, D97A, D97T, M99C, M99T, M99V, M99L, M99I, T101I, T101V, T101L, V102M, N170K, N170D, N170L, V179A, T1961, T196V, T196L, T198I, T198V, T198L, S199Y, S199F, L200P, L200C, L200S, L200A, S207R, S207K, S207M, H212R, H212K, E213K, E213R, F214A, F214G, F214S, L215P, L215Q, L215N, L215D, L215T, L215S, L215G, L215A, L215C, L215F, L215M, H221R and H221K, or a combination thereof.

3. The variant of claim 1 , comprising a mutation selected from T198I, F214S, V179A, M99I, L200P and L215P.

4. A nucleic acid sequence encoding the variant of E. coli GCH1 of claim 1 .

5. A recombinant microbial cell comprising the variant of claim 1 .

6. The recombinant microbial cell of claim 5 , further comprising nucleic acid sequences encoding a PCD and a monooxygenase.

7. The recombinant microbial cell of claim 6 , wherein
(a) The recombinant microbial cell of claim 6 , wherein
(a) the PCD is derived from Chromobacterium violecum, Homo sapiens, Pseudomonas aeruginosa and Rattus norvegicus ; or a functionally active variant thereof;
(b) the monooxygenase is a TPH derived from a Schistosoma mansoni, Homo sapiens, Gallus gallus, Bos taurus, Sus scrofa, Equus caballus, Mus musculus and Oryctolagus cuniculus TPH; or a functionally active variant thereof;
(c) the monooxygenase is a TH derived from Rattus norwegicus, Homo sapiens, Mus musculus, Bos taurus, Gallus gallus or a functionally active variant thereof;
(d) the monooxygenase is a PheH derived from Chromobacterium violaceum, Xanthomonas campestris pv. Viticola, Pseudomonas aeruginosa, Pseudomonas putida, Homo sapiens, Mus musculus, Streptomyces coeruleorubidus or a functionally active variant thereof; or
(e) a combination of (a) and (b), (a) and (c) or (a) and (d).

8. The recombinant microbial cell of claim 6 , wherein each nucleic acid sequence is operably linked to an inducible, a regulated or a constitutive promoter, and/or wherein at least the nucleic acid sequence encoding the variant of E. coli GCH1 is chromosomally integrated.

9. The recombinant microbial cell of claim 6 , which is derived from a bacterial cell, a yeast cell, a filamentous fungal cell, or an algal cell.

10. The recombinant microbial cell of claim 9 , which is derived from an Escherichia , a Saccharomyces , a Schizosaccharomyces , a Corynebacterium , a Bacillus or a Streptomyces cell.

11. The recombinant microbial cell of claim 6 , further comprising
(a) a nucleic acid sequence encoding a 5HTP decarboxylase (ADDC);
(b) nucleic acid sequences encoding an ADDC and a serotonin acetyltransferase (AANAT);
(c) nucleic acid sequences encoding an ADDC, an AANAT, and an acetylserotonin O-methyltransferase (ASMT); or
(d) nucleic acid sequences encoding a dopa decarboxylase, a tyramine oxidase and an alcohol dehydrogenase.

12. A method of producing one or more oxidation products of an aromatic amino acid, comprising culturing the recombinant microbial cell of claim 6 in a medium comprising a carbon source, and, optionally, isolating the oxidation product.

13. The method of claim 12 , wherein the oxidation product comprises at least one of 5HTP, L-DOPA, tyrosine, m-tyrosine, hydroxytyrosol, serotonin and melatonin.

14. A variant of claim 1 , E. coli GTP cyclohydrolase I (GCH1) having at least 94% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16 and comprising an amino acid substitution selected from T1981, V179A, M99I, F214S and L200P, or a combination of amino acid substitutions selected from (a) to (n):
(a) I67V, T117I, A125D, H221R;
(b) E62K, N170K, L215P;
(c) V102M, L215P;
(d) Q157L, H212R;
(e) V28L, L215P, N222I;
(f) T108N, I133F, E213K;
(g) S5C, D57V, L215Q;
(h) H29Y, I75V, V179M;
(i) A14V, E46D, M61I, D97V;
(j) V28A, G42D, E213K;
(k) N52K, A68S, S207R;
(l) A41G, K129N, I133F;
(m) S3L, K184R, S199Y; and
(n) H12R, N170D, G187S.

15. A variant of E. coli GTP cyclohydrolase I (GCH1) having at least 98% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16,
wherein the variant comprises a mutation in amino acid residue T198, and
wherein, in an E. coli cell comprising a PCD and at least one of a TPH, a TH and a PheH, the variant provides for an increased hydroxylation activity of at least one of the TPH, TH and PheH as compared to native E. coli GCH1.

16. The variant of claim 15 , wherein the mutation is selected from T198I, T198S, T198V and T198L.

17. The variant of claim 1 , wherein the variant has at least about 98% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16.

18. The variant of claim 1 , wherein the variant provides for a hydroxylation activity of at least about 120% as compared to native E. coli GCH1 having the sequence of SEQ ID NO:16.

19. The variant of claim 2 , wherein the variant has at least about 98% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16.

20. A variant of E. coli GCH1 consisting of SEQ ID NO:16 except for a T198I mutation.

21. The recombinant microbial cell of claim 10 , wherein said Escherichia cell is an E. coli cell.

Assignees

• Univ Danmarks Tekniske

Inventors

• Luo Hao
• Förster Jochen

Classifications

• C12P13/227

  Tryptophan · CPC title

• C12N9/0071

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

• C12Y305/04016

  GTP cyclohydrolase I (3.5.4.16) · CPC title

• C12Y114/16004

  Tryptophan 5-monooxygenase (1.14.16.4) · CPC title

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title