Methods for conversion of the substrate specificity of desaturases
The present invention relates to methods for the conversion of the substrate specificity of desaturases. Specifically, the present invention pertains to a method for the conversion of the substrate specificity of a Δ5 and/or Δ6 desaturase to the substrate specificity of a Δ4 desaturase, the method comprising: identifying regions and/or amino acid residues which control the substrate specificity of (i) the Δ5 and/or Δ6 desaturase and (ii) the Δ4 desaturase; and replacing in the amino acid sequence of the mentioned Δ5 and/or Δ6 desaturase, the regions and/or amino acid residues which control the substrate specificity of the Δ5 and/or Δ6 desaturase, by the corresponding regions and/or amino acid residues which control the substrate specificity of the Δ4 desaturase, thereby converting the substrate specificity of the Δ5 and/or Δ6 desaturase to the substrate specificity of the Δ4 desaturase. The present invention further concerns a method for the conversion of the substrate specificity of a Δ4 desaturase to the substrate specificity of a Δ5 and/or Δ6 desaturase, the method comprising: identifying regions and/or amino acid residues which control the substrate specificity of (i) the Δ4 desaturase and (ii) the Δ5 and/or Δ6 desaturase; and replacing in the amino acid sequence of the indicated Δ4 desaturase, the regions and/or amino acid residues which control the substrate specificity of the Δ4 desaturase, by the corresponding regions and/or amino acid residues which control the substrate specificity of the Δ5 and/or Δ6 desaturase, thereby converting the substrate specificity of the Δ4 desaturase to the substrate specificity of the Δ5 and/or Δ6 desaturase. In addition, the invention encompasses desaturases with converted substrate specificity.
1. A method for the conversion of the substrate specificity of a front-end Δ5 and/or Δ6 desaturase to the substrate specificity of a front-end Δ4 desaturase, the method comprising:
a) identifying regions and/or amino acid residues which control the substrate specificity of
(i) the front-end Δ5 and/or Δ6 desaturase and (ii) the front-end Δ4 desaturase; and
b) replacing in the amino acid sequence of the front-end Δ5 and/or Δ6 desaturase referred to in step a), the regions and/or amino acid residues which control the substrate specificity of the front-end Δ5 and/or Δ6 desaturase, by the corresponding regions and/or amino acid residues which control the substrate specificity of the front-end Δ4 desaturase, thereby converting the substrate specificity of the front-end Δ5 and/or Δ6desaturase to the substrate specificity of the front-end Δ4 desaturase,
wherein the regions and/or amino acid residues which control the substrate specificity of the Δ4, Δ5 and/or Δ6 desaturase are localized within a transmembrane domain, wherein the transmembrane domain is the third putative transmembrane domain.
2. The method of claim 1 , wherein the Δ4, Δ5 and/or Δ6 desaturase have substrate specificity for both ω3 and ω6 substrates.
3. The method of claim 1 , wherein the substrate specificity of the Δ4 desaturase is for 22:5ω3 and 22:4ω6, the substrate specificity of the Δ5 desaturase is for 20:4ω3 and 20:3ω6, the substrate specificity of the Δ6 desaturase is for 18:3ω3 and 18:2ω6, and the substrate specificity of the Δ5 and/or Δ6 desaturase is for 20:4ω3, 20:3ω6, 18:3ω3 and 18:2ω6.
4. The method of claim 1 , wherein the Δ4, Δ5 and/or Δ6 desaturase is selected from:
a) a Δ4 desaturase from Monosiga brevicollis, Euglena gracilis, Emiliana huxleyi, Pavlova lutheri, Pavlova salina, Sphaeroforma arctica, Siganus canaliculatus, Thalassiosira pseudonana , or Thraustochytrium sp.;
b) a Δ6 desaturase from Atlantic salmon, Pythium irregulare, Mortierella alpina, Ostreococcus lucimarinus, Ostreococcus tauri, Siganus canaliculatus , or Primula farinosa;
c) a Δ5 desaturase from Thraustochytrium sp., Pavlova salina, Mortierella alpina, Atlantic salmon or Siganus canaliculatus ; and
d) a Δ5/Δ6 desaturase from Siganus canaliculatus or zebrafish.
5. The method of claim 4 , wherein the Δ4, Δ5 and/or Δ6 desaturase is a Siganus canaliculatus Δ4 desaturase comprising the amino acid sequence of SEQ ID NO: 174 and a Siganus canaliculatus Δ5/Δ6 desaturase comprising the amino acid sequence of SEQ ID NO: 172.
6. The method of claim 1 , wherein the regions and/or amino acid residues which control the substrate specificity of the Δ4 desaturase comprises the amino acid sequence “YNYN” corresponding to position 280-283 in the amino acid sequence of SEQ ID NO: 174 and the regions and/or amino acid residues which control the substrate specificity of the Δ5 and/or Δ6 desaturase comprises the amino acid sequence “FHYQ” corresponding to position 278-281 of SEQ ID NO: 172.
7. The method of claim 1 , wherein the amino acid sequences of the Δ4 desaturase and the Δ5 and/or Δ6 desaturase have a sequence identity of at least 40%.