Variant type tetraprenyl-β-curcumene cyclase and method for producing ambrein

Patent metadata

Field	Value
Publication number	US-10844407-B2
Application number	US-201716080928-A
Country	US
Kind code	B2
Filing date	Mar 3, 2017
Priority date	Mar 4, 2016
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

An object of the present invention is to provide a method for preparing ambrein, which can easily obtain the ambrein. The object can be solved by a mutated tetraprenyl-β-curcumene cyclase wherein a fourth amino acid residue of a DXDD motif, aspartic acid, is substituted with an amino acid other than aspartic acid, (a) having a QXXXGX(W/F) motif, and a QXXXX(G/A)X(F/W/Y) motif on the N-terminal side, and a QXXXGX(F/W/Y) motif, and a QXXXGXW motif and a QXXXGX(F/W) motif on the C-terminal side, and not having a QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side, with respect to the DXDD motif, (b) having 40% or more identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, (c) exhibiting ambrein production activity using squalene as a substrate.

First claim

The invention claimed is:

1. A mutated tetraprenyl-β-curcumene cyclase, comprising a polypeptide wherein the polypeptide comprises
90% or more sequence identity with an amino acid sequence of SEQ ID NO: 1 wherein aspartic acid at position 373 from an N-terminal in said amino acid sequence of SEQ ID NO: 1 is substituted with cysteine or glycine, or 90% or more sequence identity with an amino acid sequence of SEQ ID NO: 13 wherein aspartic acid at position 378 from an N-terminal in said amino acid sequence of SEQ ID NO: 13 is substituted with cysteine or glycine, and
wherein the polypeptide exhibits ambrein production activity using squalene as a substrate.

2. The mutated tetraprenyl-β-curcumene cyclase according to claim 1 , comprising the amino acid sequence of SEQ ID NO: 1, wherein aspartic acid at position 373 from the N-terminal is substituted with cysteine or glycine, or the amino acid sequence of SEQ ID NO: 13, wherein aspartic acid at position 378 from the N-terminal is substituted with cysteine or glycine.

3. A polynucleotide encoding the mutated tetraprenyl-β-curcumene cyclase according to claim 1 .

4. A microorganism comprising the polynucleotide according to claim 3 .

5. The microorganism according to claim 4 , further comprising a polynucleotide encoding hydroxymethylglutaryl CoA reductase.

6. A vector comprising a DNA having the polynucleotide according to claim 3 .

7. A cell transformed with the vector according to claim 6 .

8. The cell according to claim 7 , further comprising a vector comprising a DNA having a polynucleotide encoding hydroxymethylglutaryl CoA reductase.

9. A method for preparing ambrein comprising bringing into contact the mutated tetraprenyl-β-curcumene cyclase according to claim 1 with squalene, to obtain ambrein.

10. A method for preparing ambrein comprising culturing the microorganism according to claim 4 .

11. The mutated tetraprenyl-β-curcumene cyclase of claim 1 , comprising the amino acid sequence of SEQ ID NO: 1, wherein aspartic acid at position 373 from the N-terminal is substituted with cysteine or glycine.

12. A mutated tetraprenyl-β-curcumene cyclase of claim 1 , comprising the amino acid sequence of SEQ ID NO: 13, wherein aspartic acid at position 378 from the N-terminal is substituted with cysteine or glycine.

13. A method for preparing ambrein characterized by bringing into contact the mutated tetraprenyl-β-curcumene cyclase according to claim 1 with 8a-hydroxypolypoda-13,17,21-triene, to obtain ambrein.

14. A method for preparing 3-deoxyachilleol A characterized by bringing into contact the mutated tetraprenyl-β-curcumene cyclase according to claim 1 with squalene, to obtain 3-deoxyachilleol A.

15. A mutated tetraprenyl-β-curcumene cyclase, comprising amino acid sequence SEQ ID NO: 17 wherein aspartic acid at position 340 from an N-terminal in said amino acid sequence of SEQ ID NO: 17 is substituted with cysteine or glycine.

16. The mutated tetraprenyl-β-curcumene cyclase of claim 15 , wherein the polypeptide exhibits ambrein production activity using squalene as a substrate.

Assignees

• Univ Niigata
• Adeka Corp

Inventors

• Sato Tsutomu
• OKUNO Kotone
• Takehana Toshihiko
• Koike Seiji

Classifications

• C12Y402/01137

  Sporulenol synthase (4.2.1.137) · CPC title

• C12Y101/01034

  Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34) · CPC title

• C12P7/04 Primary

  acyclic · CPC title

• C12P7/02

  containing a hydroxy group · CPC title

• C12N9/90

  Isomerases (5.) · CPC title