The invention described materials and methods for producing oxidized 13R-MO, such as forskolin. In particular, the invention describes P450s involved in oxidation of 13R-MO including CYP76AH8, CYP76AH11, CYP76AH15, CYP76AH17, CYP71D381 and CYP76AH9. Host organisms expressing one or more of these P45
The invention described materials and methods for producing oxidized 13R-MO, such as forskolin. In particular, the invention describes P450s involved in oxidation of 13R-MO including CYP76AH8, CYP76AH11, CYP76AH15, CYP76AH17, CYP71D381 and CYP76AH9. Host organisms expressing one or more of these P450s are useful in the production of oxidized 13R-MO.
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1. A method of producing an oxidised 13R-manoyl oxide (13R-MO), comprising: (a) providing a host organism, comprising a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-manoyl oxide (13R-MO) and/or an oxidised 13R-MO derivative at the 11-position, wherein the o
1. A method of producing an oxidised 13R-manoyl oxide (13R-MO), comprising: (a) providing a host organism, comprising a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-manoyl oxide (13R-MO) and/or an oxidised 13R-MO derivative at the 11-position, wherein the oxidised 13R-MO carries a —H at the 11-position; and/or catalysing oxidation of a hydroxyl group to form an oxo-group at the 11-position of 11-hydroxyl-13R-MO and/or an oxidised 11-hydroxyl-13R-MO,wherein the enzyme comprises:(i) CYP76AH8 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;(ii) CYP76AH17 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:10;(iii) CYP76AH15 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; and/or(iv) CYP76AH11 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2;(b) incubating the host organism in the presence of 13R-MO under conditions allowing growth of the host organism; and(c) optionally isolating oxidised 13R-MO from the host organism. 2. The method of claim 1, wherein the oxidised 13-R-MO is a compound of formula I: wherein R1, R2, R3 and R4 are independently —H, —OH, or —OR, andwherein R is acyl. 3. The method of claim 1, wherein the oxidised 13R-MO is a compound of formula (II): wherein R1, R2, R3 and R4 are independently —H, —OH, or —OR, andwherein R is acyl. 4. The method of claim 2, wherein: R1 is —H or —OH;R2 is —OR or —OH, and wherein R is acyl;R3 is —OR or —OH, and wherein R is acyl; andR4 is —H or —OH. 5. The method of claim 3, wherein: R1 is —H or —OH;R2 is —OR or —OH, and wherein R is acyl;R3 is —OR or —OH, and wherein R is acyl; andR4 is —H or —OH. 6. The method of claim 1, wherein the heterologous nucleic acid encodes a polypeptide capable of: (a) catalysing hydroxylation of 13R-manoyl oxide (13R-MO) and/or an oxidised 13R-MO at the 11-position, wherein the oxidised 13R-MO carries a —H at the 11-position; and/or(b) catalysing oxidation of a hydroxyl group at the 11-position of 11-hydroxyl-13R-manoyl oxide and/or an oxidised 11-hydroxyl-13R-MO,wherein the enzyme comprises: (i) CYP76AH8 having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1;(ii) CYP76AH17 having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:10; and(iii) CYP76AH15 having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:11. 7. The method of claim 1, wherein the host organism further comprises one or more of: (a) a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-MO and/or oxidised 13R-MO at the 1-position, wherein the oxidised 13R-MO carries a —H at the 1-position;(b) a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-MO and/or oxidised 13R-MO at the 6-position, wherein the oxidised 13R-MO carries a —H at the 6-position;(c) a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-MO and/or oxidised 13R-MO at the 7-position, wherein the oxidised 13R-MO carries a —H at the 7-position; or(d) a heterologous nucleic acid encoding an enzyme capable of catalysing hydroxylation of 13R-MO and/or oxidised 13R-MO at the 9-position, wherein the oxidised 13R-MO carries a —H at the 9-position,wherein at least one of the heterologous nucleic acids encodes a polypeptide capable of catalysing hydroxylation of 11-keto-13R-manoyl oxide (13R-MO) and/or an oxidised 11-keto-13R-MO derivative at one or more of the positions 1, 6, 7 and/or 9, wherein the polypeptide is CYP76AH11 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. 8. The method of claim 1, wherein the host organism is a microorganism. 9. The method of claim 8, wherein the microorganism is yeast. 10. The method of claim 1, wherein the host organism is a plant. 11. The method of claim 1, wherein the host organism further comprises a heterologous nucleic acid encoding TPS2 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. 12. The method of claim 1, wherein the host organism further comprises a heterologous nucleic acid encoding TPS3 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8 or TPS4 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.
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이 특허에 인용된 특허 (4)
Lundquist Ronald C. ; Walters David A., Fertile transgenic corn plants.
Sanford John C. (Geneva) DeVit Michael J. (Geneva NY) Bruner Ronald F. (Sewell NJ) Johnston Stephen A. (Durham NC), Method and apparatus for introducing biological substances into living cells.
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