Tyrosine hydroxylase variants and methods of use thereof

Patent metadata

Field	Value
Publication number	US-10457918-B2
Application number	US-201515507711-A
Country	US
Kind code	B2
Filing date	Sep 24, 2015
Priority date	Sep 26, 2014
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

Abstract

The present disclosure provides a variant tyrosine hydroxylase that provides for increased production of L-DOPA in a host cell that expresses the tyrosine hydroxylase. The present disclosure provides nucleic acids encoding the variant tyrosine hydroxylase, and host cells genetically modified with the nucleic acids. The present disclosure provides methods of making L-DOPA in a host cell. The present disclosure provides methods of making a benzylisoquinoline alkaloid (BIA), or a BIA precursor. The present disclosure provides methods of detecting L-DOPA level in a cell. The present disclosure provides methods of identifying tyrosine hydroxylase variants that provide for increased L-DOPA production; and methods of identifying gene products that provide for increased tyrosine production.

First claim

What is claimed is:

1. A method of producing L-3,4-dihydroxyphenylalanine (L-DOPA), the method comprising:
culturing a host cell to thereby produce L-DOPA,
wherein the host cell is genetically modified to express a heterologous tyrosine hydroxylase, wherein the heterologous tyrosine hydroxylase comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1,
wherein the tryptophan corresponding to amino acid 13 of SEQ ID NO: 1 is replaced with a different amino acid in the heterologous tyrosine hydroxylase, and/or
the phenylalanine corresponding to amino acid 309 of SEQ ID NO: 1 is replaced with a different amino acid in the heterologous tyrosine hydroxylase, and
wherein the heterologous tyrosine hydroxylase is produced in the host cell, and catalyzes the conversion of tyrosine to L-DOPA.

2. The method of claim 1 , wherein L-DOPA is produced in an amount of at least 5 μg per milliliter of culture.

3. A method of producing (S)-reticuline, the method comprising:
culturing a host cell to thereby produce (S)-reticuline,
wherein the host cell is genetically modified to express a heterologous tyrosine hydroxylase, wherein the heterologous tyrosine hydroxylase comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the heterologous tyrosine hydroxylase is produced in the host cell, and catalyzes the conversion of tyrosine to L-DOPA;
wherein the host cell is genetically modified to express a heterologous DOPA decarboxylase (DODC), wherein the heterologous DODC catalyzes the conversion of L-DOPA to dopamine,
wherein the host cell comprises endogenous enzymes that convert tyrosine to 4-hydroxyphenylacetaldehyde and the host cell converts tyrosine to 4-hydroxyphenylacetaldehyde,
wherein the host cell is genetically modified to express a heterologous norcoclaurine synthase (NCS), and wherein the heterologous NCS condenses the 4-hydroxyphenylacetaldehyde with the dopamine to produce (S)-norcoclaurine, and
wherein the host cell is genetically modified to express heterologous norcoclaurine 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT), N-methylcoclaurine hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase (4′OMT), and wherein the heterologous 6OMT, CNMT, NMCH, and 4′ OMT convert (S)-norcoclaurine to (S)-reticuline.

4. The method of claim 3 , further comprising purifying the (S)-reticuline.

5. The method of claim 3 , wherein the NCS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25 and SEQ ID NO: 26.

6. A method of producing (S)-norcoclaurine, the method comprising:
culturing a host cell to thereby produce (S)-nococlaurine,
wherein the host cell is genetically modified to express a heterologous tyrosine hydroxylase, wherein the heterologous tyrosine hydroxylase comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the heterologous tyrosine hydroxylase is produced in the host cell, and catalyzes the conversion of tyrosine to L-DOPA;
wherein the host cell is genetically modified to express a heterologous DOPA decarboxylase (DODC), wherein the heterologous DODC catalyzes the conversion of L-DOPA to dopamine,
wherein the host cell comprises endogenous enzymes that convert tyrosine to 4-hydroxyphenylacetaldehyde and the host cell converts tyrosine to 4-hydroxyphenylacetaldehyde, and
wherein the host cell is genetically modified to express a heterologous norcoclaurine synthase (NCS), and wherein the heterologous NCS condenses the 4-hydroxyphenylacetaldehyde with the dopamine to produce (S)-norcoclaurine.

7. The method of claim 1 , wherein the tryptophan corresponding to amino acid 13 of SEQ ID NO: 1 is replaced with leucine, isoleucine, or valine.

8. The method of claim 1 , wherein the phenylalanine corresponding to amino acid 309 of SEQ ID NO: 1 is replaced with leucine, isoleucine, or valine.

Assignees

• Univ California
• Valorbec S E C

Inventors

• Martin Vincent J J
• Narcross Lauren
• Dueber John E
• Deloache William C
• Russ Zachary N
• Scrivens P James

Classifications

• C12N9/0071 Primary

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

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12P13/225

  Tyrosine; 3,4-Dihydroxyphenylalanine · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• C12P7/24

  containing a carbonyl group · CPC title