Pterin-dependent biocatalysts and uses thereof

Patent metadata

Field	Value
Publication number	US-12319948-B2
Application number	US-202117381553-A
Country	US
Kind code	B2
Filing date	Jul 21, 2021
Priority date	Jan 2, 2015
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

Abstract

Provided herein are biocatalysts and systems thereof for pterin-dependent enzymes and pathways and methods of making and using the same.

First claim

We claim:

1. A biocatalyst comprising:
a tetrahydrobiopterin source, wherein the tetrahydrobiopterin source is a tetrahydrobiopterin synthesis pathway comprising:
a GTP cyclohydrase, wherein the GTP cyclohydrolase is encoded by a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4;
a pyruvoyl tetrahydropterin synthase, wherein the pyruvoyl tetrahydropterin synthase is encoded by a nucleotide sequence comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and
a sepiapterin reductase, wherein the sepiapterin reductase is encoded by a nucleotide sequence comprising SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; and

a pterin-dependent enzymatic pathway, wherein the pterin-dependent enzymatic pathway comprises tryptophan hydroxylase and aromatic-l-amino acid decarboxylase wherein the aromatic-1-amino acid decarboxylase is encoded by a nucleotide sequence comprising SEQ ID NO:17.

2. The biocatalyst of claim 1 , further comprising a synthase.

3. The biocatalyst of claim 2 , wherein the synthase is a terpene alkaloid synthase, strictosidine synthase, or a deacetylisoipecoside synthase.

4. The biocatalyst of claim 1 , further comprising a tetrahydrobiopterin recycling pathway, the tetrahydrobiopterin recycling pathway comprising:
a pterin-4a-carbinolamine dehydratase; and
a dihydropterin reductase.

5. An engineered cell comprising:
a biocatalyst, the biocatalyst comprising:
a tetrahydrobiopterin source, wherein the tetrahydrobiopterin source is a tetrahydrobiopterin synthesis pathway comprising:
a GTP cyclohydrase, wherein the GTP cyclohydrolase is encoded by a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4;
a pyruvoyl tetrahydropterin synthase, wherein the pyruvoyl tetrahydropterin synthase is encoded by a nucleotide sequence comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and
a sepiapterin reductase, wherein the sepiapterin reductase is encoded by a nucleotide sequence comprising SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; and

a pterin-dependent enzymatic pathway, wherein the pterin-dependent enzymatic pathway comprises tryptophan hydroxylase and aromatic-l-amino acid decarboxylase wherein the aromatic-1-amino acid decarboxylase is encoded by a nucleotide sequence comprising SEQ ID NO:17.

6. The engineered cell of claim 5 , further comprising a synthase.

7. The engineered cell of claim 6 , wherein the synthase is a terpene alkaloid synthase or a strictosidine synthase.

8. The engineered cell of claim 5 , further comprising a tetrahydrobiopterin recycling pathway, the tetrahydrobiopterin recycling pathway comprising:
a pterin-4a-carbinolamine dehydratase; and
a dihydropterin reductase.

9. The biocatalyst of claim 1 , wherein the amino acid mono-oxygenase is encoded by a nucleotide sequence comprising SEQ ID NO: 15.

10. The engineered cell of claim 5 , wherein the amino acid mono-oxygenase is encoded by a nucleotide sequence comprising SEQ ID NO: 15.

11. A biocatalyst comprising:
a tetrahydrobiopterin source;
a pterin-dependent enzymatic pathway, wherein the pterin-dependent enzymatic pathway comprises tryptophan hydroxylase encoded by a nucleotide sequence comprising SEQ ID NO: 15 and aromatic-l-amino acid decarboxylase; and
a tetrahydrobiopterin recycling pathway, wherein the tetrahydrobiopterin recycling pathway comprises a pterin-4a-carbinolamine dehydratase and a dihydropterin reductase.

12. The biocatalyst of claim 11 , wherein the aromatic-l-amino acid decarboxylase is encoded by a nucleotide sequence comprising SEQ ID NO: 17.

13. The biocatalyst of claim 11 , wherein the pterin-4a-carbinolamine dehydratase is encoded by a nucleotide sequence comprising SEQ ID NO: 12 and the dihydropterin reductase is encoded by a nucleotide sequence comprising SEQ ID NO: 13.

14. The biocatalyst of claim 11 , wherein the tetrahydrobiopterin source is a tetrahydrobiopterin synthesis pathway comprising:
a GTP cyclohydrase encoded by a nucleotide sequence comprising SEQ ID NO: 1;
a pyruvoyl tetrahydropterin synthase encoded by a nucleotide sequence comprising SEQ ID NO: 5; and
a sepiapterin reductase encoded by a nucleotide sequence comprising SEQ ID NO: 9.

Assignees

• Georgia Tech Res Inst

Inventors

• Peralta-Yahya Pamela
• Ehrenworth Amy M

Classifications

• C12Y402/03012

  6-Pyruvoyltetrahydropterin synthase (4.2.3.12) · CPC title

• C12Y402/01096

  4a-Hydroxytetrahydrobiopterin dehydratase (4.2.1.96) · CPC title

• C12Y401/01028

  Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase · CPC title

• C12Y305/04016

  GTP cyclohydrolase I (3.5.4.16) · CPC title

• C12Y106/99

  with other acceptors (1.6.99) · CPC title