Fungal delta 12 desaturase and delta 15 desaturase motifs

Abstract

The present invention relates to fungal Δ12 desaturases (responsible for conversion of oleic acid to linoleic acid (18:2, LA)) and Δ15 fatty acid desaturases (responsible for conversion of LA to α-linolenic acid (18:3, ALA)). Amino acid motifs diagnostic of Δ12 desaturases and Δ15 desaturases are also provided. Methods of altering enzyme specificity towards Δ12 desaturation or towards Δ15 desaturation and/or increasing production of specific ω-3 and ω-6 fatty acids by over-expression of the fungal desaturases are also described.

1 . A fungal Δ12 desaturase motif having an amino acid sequence as set forth in SEQ ID NO:5. 
     
     
         2 . The motif of  claim 1  having an amino acid sequence as set forth in SEQ ID NO:4. 
     
     
         3 . The motif of  claim 1  having an amino acid sequence as set forth in SEQ ID NO:3. 
     
     
         4 . A fungal Δ15 desaturase motif having an amino acid sequence as set forth in SEQ ID NO:48. 
     
     
         5 . The motif of  claim 4  having an amino acid sequence as set forth in SEQ ID NO:47. 
     
     
         6 . The motif of  claim 4  having an amino acid sequence as set forth in SEQ ID NO:46. 
     
     
         7 . A nucleic acid molecule encoding the fungal Δ12 desaturase motif of  claim 1 . 
     
     
         8 . A nucleic acid molecule encoding the fungal Δ15 desaturase motif of  claim 4 . 
     
     
         9 . A method for identifying a fungal polypeptide having Δ12 desaturase activity from a pool of Δ12/Δ15 desaturase-like polypeptides comprising:
 a) identifying a fungal Δ12/Δ15 desaturase-like polypeptide; and,   b) confirming the presence of a Δ12 desaturase motif in the Δ12/Δ15 desaturase-like polypeptide, wherein the Δ12 desaturase motif is selected from the group consisting of SEQ ID NOs:3, 4 and 5, and wherein the presence of the Δ12 desaturase motif is indicative of Δ12 desaturase activity.   
     
     
         10 . The method of  claim 9  wherein the fungal Δ12/Δ15 desaturase-like polypeptide has either Δ12 desaturase activity or Δ15 desaturase activity. 
     
     
         11 . The method of  claim 9  wherein the fungal Δ12/Δ15 desaturase-like polypeptide has at least 27.3% identity with a known fungal Δ15 desaturase based on the Clustal W method of alignment, using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         12 . The method of  claim 9  wherein the fungal Δ12/Δ15 polypeptide has at least 24.0% identity with a known fungal Δ12 desaturase based on the Clustal W method of alignment, using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         13 . A method for identifying a fungal polypeptide having Δ15 desaturase activity from a pool of Δ12/Δ15 desaturase-like polypeptides comprising:
 a) identifying a fungal Δ12/Δ15 desaturase-like polypeptide; and   b) confirming the presence of a Δ15 desaturase motif in the Δ12/Δ15 desaturase-like polypeptide, wherein the Δ15 desaturase motif is selected from the group consisting of SEQ ID NOs:46, 47 and 48, and wherein the presence of the Δ15 desaturase motif is indicative of Δ15 desaturase activity.   
     
     
         14 . The method of  claim 13  wherein the fungal Δ12/Δ15 desaturase-like polypeptide has either Δ12 or Δ15 desaturase activity. 
     
     
         15 . The method of  claim 13  wherein the fungal Δ12/Δ15 desaturase-like polypeptide has at least 31.8% identity with a known fungal Δ15 desaturase based on the Clustal W method of alignment, using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         16 . The method of  claim 13  wherein the fungal Δ12/Δ15 desaturase-like polypeptide of fungal origin has at least 27.3% identity with a known fungal Δ12 desaturase based on the Clustal W method of alignment, using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         17 . An isolated fungal polypeptide having Δ   12   desaturase activity isolated by the method of  claim 9 , excluding those Δ12 desaturase polypeptides isolated from species selected from the following:  Saccharomyces kluyveri, Mortierella alpina, Fusarium graminearum, Fusarium moniliforme, Magnaporthe grisea, Neurospora crassa, Aspergillus nidulans, Mortierella isabellina, Pichia pastoris, Aspergillus parasiticus, Cryptococcus curvatus, Saprolegnia diclina, Yarrowia lipolytica, Lentinula edodes, Mucor circinelloides, Mucor rouxii, Rhizopus oryzae, Botrytis cinerea  and  Aspergillus flavus.    
     
     
         18 . An isolated fungal polypeptide having Δ15 desaturase activity isolated by the method of  claim 13 , excluding those Δ15 desaturase polypeptides isolated from species selected from the following:  Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans, Neurospora crassa, Fusarium graminearum, Fusarium moniliforme  and  Magnaporthe grisea.    
     
     
         19 . A method of obtaining a nucleic acid fragment encoding a Δ12 desaturase enzyme comprising:
 a) probing a fungal genomic library with an isolated nucleic acid molecule encoding a Fungal Δ12 Desaturase Motif selected from the group consisting of SEQ ID NOs:3, 4 and 5;   b) identifying a DNA clone that hybridizes with the isolated nucleic acid molecule encoding a Fungal Δ12 Desaturase Motif of step (a); and,   c) sequencing the genomic fragment that comprises the clone identified in step (b);   wherein the sequenced genomic fragment encodes a Δ12 desaturase enzyme.   
     
     
         20 . A method of obtaining a nucleic acid fragment encoding a Δ12 desaturase enzyme comprising:
 a) providing a fungal genomic library;   b) synthesizing at least one oligonucleotide primer corresponding to a portion of an isolated nucleic acid molecule encoding a Fungal Δ12 Desaturase Motif selected from the group consisting of SEQ ID NOs:3, 4 and 5; and,   c) performing primer directed amplification on the genomic library of (a) with the primers of (b) to obtain amplification products wherein the amplification products encode a portion of an amino acid sequence encoding a Δ12 desaturase enzyme.   
     
     
         21 . A method of obtaining a nucleic acid fragment encoding a Δ15 desaturase enzyme comprising:
 a) probing a fungal genomic library with an isolated nucleic acid molecule encoding a Fungal Δ15 Desaturase Motif selected from the group consisting of SEQ ID NOs:46, 47 and 48;   b) identifying a DNA clone that hybridizes with the isolated nucleic acid molecule encoding a Fungal Δ15 Desaturase Motif of step (a); and,   c) sequencing the genomic fragment that comprises the clone identified in step (b) wherein the sequenced genomic fragment encodes a Δ15 desaturase enzyme.   
     
     
         22 . A method of obtaining a nucleic acid fragment encoding a Δ15 desaturase enzyme comprising:
 a) providing a fungal genomic library;   b) synthesizing at least one oligonucleotide primer corresponding to a portion of an isolated nucleic acid molecule encoding a Fungal Δ15 Desaturase Motif selected from the group consisting of SEQ ID NOs:46, 47 and 48; and,   c) performing primer directed amplification on the genomic library of (a) with the primers of (b) to obtain amplification products wherein the amplification products encode a portion of an amino acid sequence encoding a Δ15 desaturase enzyme.   
     
     
         23 . An isolated fungal nucleic acid molecule encoding a polypeptide having Δ12 desaturase activity isolated by the method of either of  claims 19  or  20 , excluding nucleic acid molecules having Δ12 desaturase activity isolated from species selected from the following:  Saccharomyces kluyveri, Mortierella alpina, Fusarium graminearum, Fusarium moniliforme, Magnaporthe grisea, Neurospora crassa, Aspergillus nidulans, Mortierella isabellina, Pichia pastoris, Aspergillus parasiticus, Cryptococcus curvatus, Saprolegnia diclina, Yarrowia lipolytica, Lentinula edodes, Mucor circinelloides, Mucor rouxii, Rhizopus oryzae, Botrytis cinerea  and  Aspergillus flavus.    
     
     
         24 . An isolated fungal nucleic acid molecule encoding a polypeptide having Δ15 desaturase activity isolated by the method of either of  claims 21  or  22  excluding nucleic acid molecules having Δ15 desaturase activity isolated from species selected from the following:  Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans, Neurospora crassa, Fusarium graminearum, Fusarium moniliforme  and  Magnaporthe grisea.    
     
     
         25 . A method for increasing the ability of a bifunctional fungal Δ15 desaturase enzyme to act on a Δ12 desaturase substrate comprising:
 a) providing a nucleic acid molecule encoding a polypeptide having bifunctional Δ15 desaturase activity and comprising a Δ15 desaturase motif having an amino acid sequence selected from the group consisting of SEQ ID NOs:46, 47 and 48, wherein each of the motif sequences comprises an isoleucine at amino acid residue 4; and,   b) altering the nucleic acid molecule of (a) such that it encodes a mutant polypeptide comprising a mutant Δ15 desaturase motif of (a) wherein the isoleucine at amino acid residue 4 is replaced with valine, wherein the mutant polypeptide has an increased ability to act on a Δ12 desaturase substrate.   
     
     
         26 . A method for increasing the ability of a bifunctional fungal Δ12 desaturase enzyme to act on a Δ15 desaturase substrate comprising:
 a) providing a nucleic acid molecule encoding a polypeptide having bifunctional Δ12 desaturase activity and comprising a Δ12 desaturase motif having an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 4 and 5, wherein each of the motif sequences comprises a valine at amino acid residue 4; and,   b) altering the nucleic acid molecule of (a) such that it encodes a mutant polypeptide comprising a mutant Δ12 desaturase motif of (a) wherein the valine at amino acid residue 4 is replaced with isoleucine, wherein the mutant polypeptide has an increased ability to act on a Δ15 desaturase substrate.   
     
     
         27 . A method for the production of linoleic acid comprising:
 a) providing a host cell comprising:
 i) an isolated nucleic acid fragment encoding a polypeptide having Δ12 desaturase activity and comprising a Δ12 desaturase motif having an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 4 and 5; and, 
 ii) a source of oleic acid; 
   b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding the desaturase polypeptide is expressed and the oleic acid is converted to linoleic acid; and,   c.) optionally recovering the linoleic acid of step (b).   
     
     
         28 . A method according to  claim 27  wherein the isolated nucleic acid fragment encoding the desaturase polypeptide is selected from the group consisting of:
 a.) a polypeptide selected from the group consisting of: SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:91 and SEQ ID NO:87; and,   b.) a polypeptide whose amino acid sequence is selected from the group consisting of the following genomic sequence ORFs:
 i.) [SEQ ID NO:67]; 
 ii.) [SEQ ID NO:69]; and 
 iii.) [SEQ ID NO:71]. 
   
     
     
         29 . A method for the production of α-linolenic acid comprising:
 a.) providing a host cell comprising:
 i.) an isolated nucleic acid fragment encoding a polypeptide having Δ15 desaturase activity and comprising a Δ15 desaturase motif having an amino acid sequence selected from the group consisting of SEQ ID NOs:46, 47 and 48; and, 
 ii.) a source of linoleic acid; 
   b.) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding the desaturase polypeptide is expressed and the linoleic acid is converted to α-linolenic acid; and,   c.) optionally recovering the α-linolenic acid of step (b).   
     
     
         30 . A method according to  claim 29  wherein the isolated nucleic acid fragment encoding the desaturase polypeptide is selected from the group consisting of:
 a.) a polypeptide selected from the group consisting of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78 and SEQ ID NO:80; and,   b.) a polypeptide whose amino acid sequence is selected from the group consisting of the following genomic sequence ORFs:
 i.) [SEQ ID NO:68]; 
 ii.) [SEQ ID NO:70]; and, 
 iii.) [SEQ ID NO:72]. 
   
     
     
         31 . A method according to  claim 29  wherein the desaturase polypeptide having Δ15 desaturase activity binds both oleic acid and linoleic acid as enzymatic substrates. 
     
     
         32 . A method according to either of  claims 27  or  29  wherein the host cell is an oleaginous yeast. 
     
     
         33 . A method according to  claim 32  wherein the oleaginous yeast is selected from the group consisting of:  Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon  and  Lipomyces.    
     
     
         34 . A method according to  claim 33  wherein the  Yarrowia  is selected from the group consisting of:  Yarrowia lipolytica  ATCC #20362,  Yarrowia lipolytica  ATCC #8862,  Yarrowia lipolytica  ATCC #18944,  Yarrowia lipolytica  ATCC #76982 and  Yarrowia lipolytica  LGAM S(7)1. 
     
     
         35 . A method for identifying a polynucleotide sequence encoding a fungal polypeptide having Δ15 desaturase activity comprising:
 a) providing at least one polynucleotide sequence encoding a Δ15 desaturase motif selected from the group consisting of SEQ ID NOs:46, 47 and 48, on a computer-readable format;   b) comparing, on the computer-readable format, the at least one polynucleotide sequence of (a) with at least one polynucleotide sequence from at least one fungal genome; and,   c) identifying a fungal sequence comprising the at least one polynucleotide sequence encoding a Δ15 desaturase motif;   
       wherein the fungal sequence of (c) comprising the at least one polynucleotide sequences encoding a Δ15 desaturase motif encodes a Δ15 desaturase polypeptide having Δ15 desaturase activity. 
     
     
         36 . A method according to  claim 35  wherein the at least one polynucleotide sequence from at least one fungal genome are comprised of sequences encoding fungal Δ12/Δ15 desaturase-like polypeptides. 
     
     
         37 . A method for identifying an amino acid sequence of a fungal polypeptide having Δ15 desaturase activity comprising:
 a) providing at least one amino acid sequence of a Δ15 desaturase motif selected from the group consisting of SEQ ID NOs:46, 47 and 48, on a computer-readable format;   b) comparing, on the computer-readable format, the at least one amino acid sequence of (a) with at least one amino acid sequence from at least one fungal genome;   c) identifying a fungal sequence comprising the at least one amino acid sequence of a Δ15 desaturase motif;   
       wherein the fungal sequence of (c) comprising the at least one amino acid sequence of a Δ15 desaturase motif defines a Δ15 desaturase polypeptide having Δ15 desaturase activity. 
     
     
         38 . A method according to  claim 37  wherein the at least one amino acid sequence from at least one fungal genome are comprised of sequences defining fungal Δ12/Δ15 desaturase-like polypeptides. 
     
     
         39 . A method for identifying a polynucleotide sequence encoding a fungal polypeptide having Δ12 desaturase activity comprising:
 a) providing at least one polynucleotide sequence encoding a Δ12 desaturase motif selected from the group consisting of SEQ ID NOs:3, 4 and 5, on a computer-readable format;   b) comparing, on the computer-readable format, the at least one polynucleotide sequence of (a) with at least one polynucleotide sequence from at least one fungal genome; and,   c) identifying a fungal sequence comprising the at least one polynucleotide sequence encoding a Δ12 desaturase motif;   
       wherein the fungal sequence of (c) comprising the at least one polynucleotide sequences encoding a Δ12 desaturase motif encodes a Δ12 desaturase polypeptide having Δ12 desaturase activity. 
     
     
         40 . A method according to  claim 39  wherein the at least one polynucleotide sequence from at least one fungal genome are comprised of sequences encoding fungal Δ12/Δ15 desaturase-like polypeptides. 
     
     
         41 . A method for identifying an amino acid sequence of a fungal polypeptide having Δ12 desaturase activity comprising:
 a) providing at least one amino acid sequence of a Δ12 desaturase motif selected from the group consisting of SEQ ID NOs:3, 4 and 5, on a computer-readable format;   b) comparing, on the computer-readable format, the at least one amino acid sequence of (a) with at least one amino acid sequence from at least one fungal genome;   c) identifying a fungal sequence comprising the at least one amino acid sequence of a Δ12 desaturase motif;   
       wherein the fungal sequence of (c) comprising the at least one amino acid sequence of a Δ12 desaturase motif defines a Δ12 desaturase polypeptide having Δ12 desaturase activity. 
     
     
         42 . A method according to  claim 41  wherein the at least one amino acid sequence of at least one fungal genome are comprised of sequences defining fungal Δ12/Δ15 desaturase-like polypeptides.