Some content of this application is unavailable at the moment.
If this situation persist, please contact us atFeedback&Contact
1. (WO2018104238) KAURENOIC ACID HYDROXYLASES
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

CLAIMS

1. A polypeptide having kaurenoic acid 13-hydroxylase activity, which polypeptide comprises an amino acid sequence which, when aligned with a kaurenoic acid 13-hydroxylase comprising the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 3, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions

136, 248, 336 or 403

said positions being defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3 and wherein the polypeptide has one or more modified properties as compared with a reference polypeptide having kaurenoic acid 13-hydroxylase activity.

2. A polypeptide according to claim 1 wherein the positions in the polypeptide having kaurenoic acid 13-hydroxylase activity corresponding to any amino acids at position 136, 248,

336 or 403, said position being defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3, are identified by aligning the amino acid sequence of the polypeptide with kaurenoic acid 13-hydroxylase activity with the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 3 using the EMBOSS Needle alignment method, using EBLOSUM62 as a substitution matrix, with a gap-open penalty of 10 and a gap extension penalty of 0.5.

3. A polypeptide according to claim 1 or 2, wherein the modified property is modified kaurenoic acid 13-hydroxylase activity.

4. A polypeptide according to any one of claims 1 to 3, wherein the reference polypeptide comprises the kaurenoic acid 13-hydroxylase of SEQ ID NO: 1 or SEQ ID NO: 3.

5. A polypeptide according to any one of the preceding claims, wherein:

(i) a methionine or valine is present at position 136;

(ii) an asparagine is present at position 248;

(iii) a serine is present at position 336; and/or

(iv) a glycine is present at position 403,

said positions being defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3.

6. A polypeptide according to any one of the preceding claims comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3.

7. A polypeptide having kaurenoic acid 13-hydroxylase activity comprising an amino acid sequence having at least about 95% sequence identity, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 5, 7, 9, 1 1 or 13.

8. A nucleic acid comprising a sequence encoding a polypeptide according to any one of the preceding claims.

9. A recombinant host comprising a nucleic acid according to claim 8, optionally which is capable of producing steviol or a steviol glycoside.

10. A recombinant host according to claim 9 which comprises one or more recombinant nucleotide sequence(s) encoding:

a polypeptide having ent-copalyl pyrophosphate synthase activity;

a polypeptide having ent-Kaurene synthase activity; and

a polypeptide having ent-Kaurene oxidase activity; and, optionally,

a polypeptide having kaurenoic acid 13-hydroxylase activity which is different from a polypeptide according to any one of claims 1 to 6.

1 1. A recombinant host according to claim 9 or 10, which comprises a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

12. A recombinant host according to any one of claims 9 to 1 1 which comprises a recombinant nucleic acid sequence encoding one or more of:

(i) a polypeptide having UGT74G1 activity;

(ii) a polypeptide having UGT2 activity;

(iii) a polypeptide having UGT85C2 activity; and

(iv) a polypeptide having UGT76G1 activity.

13. A recombinant host according to any one of claims 9 to 12, wherein the host belongs to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia, for example a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell or an Escherichia coli cell.

14. A process for the preparation of steviol or a steviol glycoside which comprises fermenting a recombinant host according to any one of claims 9 to 13 in a suitable fermentation medium and, optionally, recovering the steviol or steviol glycoside.

15. A fermentation broth comprising a steviol glycoside obtainable by the process according to claim 14.

16. A composition comprising one or more steviol glycosides obtained by a process according to claim 14 or obtained from a fermentation broth according to claim 15.

17. A foodstuff, feed or beverage which comprises a composition according to claim 16.

18. A method for converting steviol or a first steviol glycoside into a second steviol glycoside, which method comprises:

contacting steviol or first steviol glycoside with a recombinant host according to any one of claims 9 to 13, a cell free extract derived from such a recombinant host or an enzyme preparation derived from either thereof;

thereby to convert the steviol or first steviol glycoside into the second steviol glycoside.