US2015275191A1PendingUtilityA1

Triterpene oxidase derived from plant belonging to genus glycyrrhiza, gene encoding the same, and method of using the same

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Assignee: RIKENPriority: Aug 29, 2008Filed: Jun 15, 2015Published: Oct 1, 2015
Est. expiryAug 29, 2028(~2.1 yrs left)· nominal 20-yr term from priority
Inventors:Toshiya MuranakaHikaru SekiKiyoshi OhyamaHiroshi SudoSatoru SawaiKazuki Saito
C12Y 114/13134C12P 15/00C12N 9/0073C12P 7/40C12N 9/0006C12P 33/00
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Claims

Abstract

Identification of a protein having an activity of oxidizing oleanane-type triterpene, and a gene encoding the protein, the protein and the gene, and use thereof are provided. For example, a protein having an activity of oxidizing oleanane-type triterpene obtained from a plant in the family Fabaceae, a gene encoding the protein and use thereof are provided. The protein is shown in, for example, SEQ ID NO: 4, 14 or 18, and the gene encoding the protein is shown in, for example, SEQ ID NO: 3, 13 or 17. A transformant into which the gene is introduced can be produced, and thereby a triterpene oxidase can be obtained.

Claims

exact text as granted — not AI-modified
1 . A polypeptide having an activity of oxidizing carbon at a position 30 of oleanane-type triterpene, wherein the isolated polypeptide comprises:
 an amino acid sequence of SEQ ID NO:4, SEQ ID NO: 14 or SEQ ID NO:18; or   an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO:4, SEQ ID NO:14 or SEQ ID NO:18.   
     
     
         2 . The polypeptide according to  claim 1 , wherein the oleanane-type triterpene is β-amyrin, 11-oxo-β-amyrin, or 30-hydroxy-11-oxo-β-amyrin. 
     
     
         3 . The polypeptide according to  claim 1 , being derived from a plant in the family Fabaceae. 
     
     
         4 . The polypeptide according to  claim 3 , wherein the plant in the family Fabaceae is a plant in the subfamily Faboideae, 
     
     
         5 . The polypeptide according to  claim 4 , wherein the plant in the subfamily Faboideae is a plant belonging to the genus  Glycyrrhiza  or the genus  Medicago.    
     
     
         6 . The polypeptide according to  claim 5 , wherein the plant belonging to the genus  Glycyrrhiza  is  G. uralensis  or  G. glabra.    
     
     
         7 . The polypeptide according to  claim 5 , wherein the plant belonging to the genus  Medicago  is  M. truncatula.    
     
     
         8 . The polypeptide according to  claim 1 , being a protein belonging to cytochrome P450. 
     
     
         9 . An isolated cDNA polynucleotide encoding a polypeptide having an activity of oxidizing carbon at a position 30 of an oleanane-type triterpene, wherein the isolated cDNA polynucleotide comprises a nucleotide sequence having 95% or more identity with SEQ ID NO:3, SEQ ID NO:13, or SEQ ID NO:17. 
     
     
         10 . The isolated cDNA polynucleotide according to  claim 9 , wherein the oleanane-type triterpene is β-amyrin, 11-oxo-β-amyrin, or 30-hydroxy-11-oxo-β-amyrin. 
     
     
         11 . The isolated cDNA polynucleotide according to  claim 10 , being derived from a plant in the family Fabaceae. 
     
     
         12 . The isolated cDNA polynucleotide according to  claim 12 , wherein the plant in the family Fabaceae is a plant in the subfamily Faboideae. 
     
     
         13 . The isolated cDNA polynucleotide according to  claim 12 , wherein the subfamily Faboideae is a plant belonging to the genus  Glycyrrhiza  or the genus  Medicago.    
     
     
         14 . The isolated cDNA polynucleotide according to  claim 12 , wherein the plant belonging to the genus  Glycyrrhiza  is  G. uralensis  or  G. glabra.    
     
     
         15 . The isolated cDNA polynucleotide according to  claim 12 , wherein the plant belonging to the genus  Medicago  is  M. truncatula.    
     
     
         16 . The isolated cDNA polynucleotide according to  claim 9 , wherein the isolated cDNA polynucleotide encodes a protein belonging to cytochrome P450. 
     
     
         17 . An isolated cDNA polynucleotide, comprising a nucleotide sequence having 95% or more identity with SEQ ID NO: 3, SEQ ID NO: 13, or SEQ ID NO: 17. 
     
     
         18 . A recombinant vector comprising the isolated cDNA polynucleotide according to  claim 9 . 
     
     
         19 . A transformant comprising the isolated cDNA polynucleotide according to  claim 9 . 
     
     
         20 . The transformant according to  claim 19 , wherein the transformant is a plant in the family Fabaceae. 
     
     
         21 . The transformant according to  claim 20 , wherein the plant in the family Fabaceae is a plant in the subfamily Faboideae. 
     
     
         22 . The transformant according to  claim 20 , wherein the plant in the family Fabaceae is a plant belonging to the genus  Glycyrrhiza  or the genus  Medicago.    
     
     
         23 . The transformant according to  claim 22 , wherein the plant belonging to the genus  Glycyrrhiza  is  G. uralensis  or  G. glabra.    
     
     
         24 . The transformant according to  claim 22 , wherein the plant belonging to the genus  Medicago  is  M. truncatula.    
     
     
         25 . The transformant according to  claim 19 , wherein expression of the isolated cDNA polynucleotide is enhanced. 
     
     
         26 . The transformant according to  claim 19 , wherein expression of the isolated cDNA polynucleotide is suppressed. claim 
     
     
         27 . A method for manufacturing a polypeptide, the method comprising:
 culturing or growing the transformant according to  claim 19 ; and   extracting the polypeptide from the cultured product or the grown product.   
     
     
         28 . A method for manufacturing glycyrrhetinic acid and 20-epi-glycyrrhetinic acid, the method comprising allowing the polypeptide according to  claim 1  and a polypeptide having an activity of an oxidizing carbon at a position 11 of an oleanane-type triterpene to act on a oleanane-type triterpene. 
     
     
         29 . A recombinant vector comprising the isolated cDNA polynucleotide according to  claim 17 . 
     
     
         30 . A transformant comprising the isolated cDNA polynucleotide according to  claim 17 . 
     
     
         31 . The transformant according to  claim 30 , wherein the transformant is a plant in the family Fabaceae. 
     
     
         32 . The transformant according to  claim 31 , wherein the plant in the family Fabaceae is a plant in the subfamily Faboideae. 
     
     
         33 . The transformant according to  claim 31 , wherein the plant in the family Fabaceae is a plant belonging to the genus  Glycyrrhiza  or the genus  Medicago.    
     
     
         34 . The transformant according to  claim 33 , wherein the plant belonging to the genus  Glycyrrhiza  is  G. uralensis  or  G. glabra.    
     
     
         35 . The transformant according to  claim 33 , wherein the plant belonging to the genus  Medicago  is  M. truncatula.    
     
     
         36 . The transformant according to  claim 31 , wherein expression of the isolated cDNA polynucleotide is enhanced. 
     
     
         37 . The transformant according to  claim 30 , wherein expression of the isolated cDNA polynucleotide is suppressed. claim 
     
     
         38 . A method for manufacturing a polypeptide, the method comprising:
 culturing or growing the transformant according to  claim 30 ; and   extracting the polypeptide from the cultured product or the grown product.

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