Methods for conversion of the substrate specificity of desaturases

Abstract

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.

Claims

1. A method for the conversion of the substrate specificity of a front-end Δ4 desaturase to the substrate specificity of a front-end Δ5 and/or Δ6 desaturase, the method comprising: a) identifying regions and/or amino acid residues which control the substrate specificity of (i) the front-end Δ4 desaturase and (ii) the front-end Δ5 and/or Δ6 desaturase; and b) replacing in the amino acid sequence of the front-end Δ4 desaturase referred to in step a), the regions and/or amino acid residues which control the substrate specificity of the front-end Δ4 desaturase, by the corresponding regions and/or amino acid residues which control the substrate specificity of the front-end Δ5 and/or Δ6 desaturase, thereby converting the substrate specificity of the front-end Δ4 desaturase to the substrate specificity of the front-end Δ5 and/or Δ6 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%.

Description

(1) The Figures show:

(2) FIG. 1: Alignment of deduced amino acid sequences of S. canaliculatus Δ4 desaturase (GenBank accession number: GU594278.1; SEQ ID NO: 174) and Δ5/Δ6 desaturase (GenBank accession number: EF424276.2; SEQ ID NO: 172) generated by VectorNTI software. Non-identical residues are highlighted in grey. The cytochrome b.sub.5-like domain is underlined by a waved line. The four amino acid residues that determine the functional divergence between the two genes are shown in bold. Histidine boxes are underlined by dotted lines and the putative transmembrane domains are underlined with solid lines.

(3) FIG. 2: Putative protein topology of S. canaliculatus Δ4 and Δ5/Δ6 desaturases, based on TOPCONS membrane topology prediction software. The membrane topology was generated by restraining N- and C-terminal regions to the inside (cytoplasmic) area. H denotes histidine box. Stars indicate the approximate location of the four amino acid residues involved in substrate chain-length specificity and regiospecificity.

(4) FIG. 3: Gas chromatograms of yeast cells containing an empty pYES2.1/V5-His-TOPO vector. A, no fatty acid supplied; B, DPA supplied exogenously; C, ALA supplied exogenously.

(5) FIG. 4: Gas chromatograms of yeast cells expressing S. canaliculatus wild-type and mutated desaturases. A, S. canaliculatus Δ4 desaturase (SEQ ID NO: 174); B, S. canaliculatus Δ5/Δ6 desaturase (SEQ ID NO: 172); C, chimeric S. canaliculatus desaturase Sig-3C; D, chimeric S. canaliculatus desaturase Sig-4C; E, chimeric S. canaliculatus desaturase Sig-5C (SEQ ID NO: 10); F, chimeric S. canaliculatus desaturase Sig-6C (SEQ ID NO: 2); G, chimeric S. canaliculatus desaturase Sig-17C (SEQ ID NO: 16) H, chimeric S. canaliculatus desaturase Sig-18C (SEQ ID NO: 8). Top and bottom panels show results from cultures supplied with DPA and ALA respectively.

(6) FIGS. 5 and 6: Schematic representation of S. canaliculatus Δ4 and Δ5/Δ6 chimeric gene constructs with associated conversion percentages. Substitution of domains within enzymes. FIG. 5: S. canaliculatus Δ4 sequence (SEQ ID NO: 174) is represented as a white bar (left) and S. canaliculatus Δ5/Δ6 sequence (SEQ ID NO: 172) is represented as a black bar (right).

(7) FIG. 6: Substitutions within a critical eight amino acid region. The amino acid residues shown in each chimeric gene represent the start and end of the domain substitution. The conversion percentages are the mean of three individual assays ±SD. S. canaliculatus Δ4 sequence (SEQ ID NO: 174) is represented as a white bar and S. canaliculatus Δ5/Δ6 sequence (SEQ ID NO: 172) is represented as a black bar. The bold amino acid residues denote the residues which were changed. The conversion rates are the mean of three individual assays ±SD.

(8) FIG. 7: Amino acid substitutions introduced into the region of Δ4 and Δ5/Δ6 desaturases critical for substrate specificity. Residue numbering is based on the Δ4 and Δ5/Δ6 desaturase amino acid sequences “YNYN” and “FHYQ”, respectively. Residues from the Δ4 desaturase were substituted into the corresponding position of the Δ5/Δ6 sequence, and vice versa.

(9) FIG. 8: Primers used in this study for constructing the chimeric proteins of S. canaliculatus Δ4 and Δ5/Δ6 desaturases and site-directed mutagenesis. F: forward primer; R: reverse primer.

(10) FIG. 9: Allocation of the nucleic acid sequences and amino acid sequences of the chimeric S. canaliculatus proteins to the corresponding SEQ ID NOs.

(11) FIG. 10: Alignment of the amino acid sequences of the chimeric S. canaliculatus proteins indicated in FIG. 9.

(12) The invention will now be illustrated by the following Examples which shall, however, not be construed as limiting the scope of the invention.

EXAMPLES

Example 1

(13) Growth and induction media for yeast expression were purchased from MP Biologicals (Solon, Ohio). Fatty acid (DPA.sub.ω3 and ALA) substrates were purchased from Nu-Chek Prep Inc (Elysian, Minn.). Phusion high fidelity DNA polymerase was from New England Biolabs (Ipswich, Mass.). Taq polymerase was from Invitrogen (Burlington, ON). All HPLC grade solvents were purchased from EMD Inc. (Mississauga, ON) and Fisher Scientific (Ottawa, ON). All chemicals if not mentioned otherwise, were purchased from Sigma-Aldrich (Oakville, ON).

(14) 1.1 S. canaliculatus Δ4 and Δ5/Δ6 Desaturases

(15) Sequences encoding the S. canaliculatus Δ4 desaturase (GenBank accession number: GU594278.1; nucleic acid sequence shown in SEQ ID NO: 173 and amino acid sequence shown in SEQ ID NO: 174) and the Δ5/Δ6 desaturase (GenBank accession number: EF424276.2; nucleic acid sequence shown in SEQ ID NO: 171 and amino acid sequence shown in SEQ ID NO: 172) were synthesized by Life Technologies Corporation (Burlington, ON), based on the GenBank database sequences. Synthetic genes were cloned into pYES2.1/V5-His-TOPO (Invitrogen) and sequenced, prior to being used in further experiments.

(16) 1.2 Construction of Vectors

(17) Chimeric enzymes were constructed from Δ4 and Δ5/Δ6 fatty acid desaturases by overlap extension PCR (‘sewing’ PCR), targeting regions in the open reading frame (ORF) of the desaturases. The positions for substitutions were rationally chosen by aligning S. canaliculatus Δ4 and Δ5/Δ6 desaturases to locate the areas with differences in amino acid composition. After regions of interest were identified, overlapping synthetic primers were designed so that fragments from different portions of the genes could be amplified then ‘sewn’ together to form a chimera gene or gene containing a specific site-directed mutation. The initial chimeric genes were constructed using the synthesized S. canaliculatus Δ4 and Δ5/Δ6 desaturases (Life Technologies) as the DNA template for PCR. Subsequent chimeric genes were constructed using either the S. canaliculatus Δ4 and Δ5/Δ6 desaturases or chimeric genes constructed in this study. The overlap extension PCR was performed using Phusion high fidelity DNA polymerase, a proofreading polymerase which produces blunt-ended DNA products. PCR parameters were as follows: initial denaturation for 2 min at 98° C., followed by 30 cycles of 98° C. denaturation for 30 s, 60° C. annealing for 30 s, and 72° C. extension for 90 s. After amplification, PCR products were subjected to electrophoresis on a 0.8% agarose gel. The DNA band with the appropriate size was cut from the gel and purified using an EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic Inc., Markham, ON), according to the manufacturer's instructions. Before cloning the purified DNA product into the pYES2.1/V5-His-TOPO yeast expression vector (Invitrogen), an additional post-amplification step using Taq polymerase was performed. This step added 3′ A-overhangs to the amplified DNA, which is necessary for TA cloning into the pYES2.1/V5-His-TOPO vector. The addition of a 3′ A-overhang was performed by adding 20 μL of gel purified DNA, 2.5 pL 10×PCR buffer minus Mg.sup.2+, 1.5 μL of 50 mM MgCl.sub.2, 0.2 mM dNTP and 2.5 units of Taq polymerase, followed by incubation at 72° C. for 10 min. The amplified DNA fragment was cloned into the pYES2.1/V5-His-TOPO vector according to the manufacturer's instructions, and the plasmid DNA was isolated using an EZ-10 spin column plasmid DNA miniprep kit (Bio Basic Inc.). After sequencing, plasmids were transformed into the S. cerevisiae yeast strain INVSc1 (Invitrogen) using the EasyComp transformation kit (Invitrogen).

(18) 1.3 Expression in Yeast

(19) For functional expression of the desaturases, yeast cultures were grown overnight at 30° C. in DOB-URA (yeast synthetic complete media devoid of uracil) containing 2% glucose. The OD600 of the overnight yeast cultures were measured and standardized between samples. The samples were washed with DOB-URA containing 2% galactose and expression was induced with the same media. Expression was carried out for 3 days at 20° C. in the presence of exogenousiy supplied fatty acids (250 μM DPA.sub.ω3 or ALA depending on the yeast construct).

(20) 1.4 Fatty Acid Analysis

(21) After 3 days of growth in the presence of exogenousiy supplied fatty acids, the yeast cells were collected by centrifugation at 2,800 rpm for 3 min and washed once with induction buffer (2% galactose) and washed again with sterile distilled water. Two mL of 3N-methanolic HCl was added to each sample and the samples were heated at 80° C. for 40 min, cooled at room temperature, and the hexane phase containing the fatty acid methyl esters was partitioned from the aqueous phase by the addition of 1 mL 0.9% NaCl and 2 mL hexane, followed by centrifugation. The hexane phase was transferred to glass vials and air-dried under a gentle stream of liquid nitrogen gas and was resuspended in 100 μL of hexane before being analyzed by gas chromatography (GC) from Agilent Technologies Inc. (model: 6890N, Integrated peaks calculated by Agilent ChemStation Software). Desaturation percent was calculated as:
100/(product/(substrate+product)).

Example 2

(22) 2.1 S. canaliculatus Δ4 and Δ5/Δ6 Desaturases have Similar Sequences and Structures

(23) The deduced proteins of the Δ4 desaturase (SEQ ID NO: 174) and Δ5/Δ6 desaturase (SEQ ID NO: 174) genes of S. canaliculatus have 445 and 443 amino acids respectively, with the two amino acid gap located in the N-terminal region of the alignment (FIG. 1). Based on membrane topology analysis (TOPCONS; restraining N- and C-terminal inside cytoplasmic area; FIG. 2), both the S. canaliculatus Δ4 and Δ5/Δ6 genes have four predicted transmembrane domains with two linker regions. Both desaturases have three conserved histidine boxes, and an N-terminal cytochrome b.sub.5-like domain, which are the characteristic features of a front-end microsomal desaturase (Li et al., 2010). Due to the high similarity in amino acid sequence between the two desaturases, the inventors were able to rationally design primers to produce chimeras based on aligning the open reading frame (ORF) of the S. canaliculatus genes (FIG. 1). Specific corresponding regions were selected and exchanged between the two desaturases to form chimeric proteins. Regions that were quite highly conserved, as well as less conserved regions were substituted.

(24) FIG. 8 lists the primers and corresponding SEQ ID NOs. used for constructing the chimeras and for performing site-directed mutagenesis to identify the roles of specific amino acid residues. This set of primers allowed the inventors to perform substitutions of domains distributed in the hydrophobic transmembrane domains, C-terminal and N-terminal regions and linkers between transmembrane domains, based on the topology analysis shown in FIG. 2.

(25) 2.2 A Four Amino Acid Region within the S. canaliculatus Δ4 and Δ5/Δ6 Desaturases Controls Substrate Length and Regiospecificity

(26) Both S. canaliculatus desaturases show a preference for ω3 fatty acids (Li et al., 2010). Therefore, the ω3 fatty acids DPA and ALA, which are substrates for the native Δ4- and Δ5/Δ6-desaturases respectively, were used throughout this study. As a negative control, S. ceriviseae INVSc1 cells were transformed with the empty pYES2.1/V5-His-TOPO vector. The fatty acids observed in the control included 14:0, 16:0, 16:1ω7, 18:0 and 18:1ω9 and 26:0, but neither Δ4 nor DPA.sub.ω3 activity was observed (FIG. 3). Expression of wild-type S. canaliculatus Δ4 (SEQ ID NO: 174) and Δ5/Δ6 desaturases (SEQ ID NO: 172) in yeast showed desaturase activities of 12.0±0.5% (DPA.sub.ω3) and 22.7±2.5% (ALA), respectively (FIGS. 4 and 5; Table 1). The Δ5/Δ6 desaturase did not show activity with DPA.sub.ω3, nor did the Δ4 desaturase show activity with ALA (FIGS. 4 and 5; Table 1).

(27) When the S. canaliculatus Δ4 desaturase sequence from the amino acid sequence “MPRH” onward, representing approximately the last one-tenth of the enzyme, was replaced with the Δ5/Δ6 sequence, the enzyme maintained the same substrate specificity but the conversion level dropped by approximately 40% (Sig-1C; FIG. 5 left column). The reverse construct (Sig-2C; FIG. 5 right column) maintained the substrate specificity of the Δ5/Δ6 enzyme but fatty acid conversion was reduced by approximately 30%. Since the last tenth of the enzymes do not appear to contribute to substrate specificity, chimeric proteins with domain swapping from the “PVYG” amino acid location onward, representing approximately the last third of the enzyme, were constructed (Sig-3C; FIG. 5 left column, and Sig-4C; FIG. 5 right column). No change in substrate specificities was observed between the two chimeras but the enzyme activities of Sig-3C and 4C were reduced by approximately 30% and 45%, respectively, compared to wild-type (see also Table 1).

(28) The regions from “LIPV” onward, representing approximately the last 40% of the coding regions, were exchanged in the corresponding chimeric constructs Sig-5C (SEQ ID NO: 10; FIG. 5 left column) and Sig-6C (SEQ ID NO: 2; FIG. 5 right column). The switch from Δ4 to Δ5/Δ6 sequence at “LIPV” caused Sig-5C to lose Δ4 activity but gain Δ6 activity, showing a conversion level of approximately 23% with ALA (Sig-5C; SEQ ID NO: 10; FIG. 5 left column; Table 1). Conversely, the reverse construct lost 46 activity but gained activity with DPA (Sig-6C; SEQ ID NO: 2; FIG. 5 right column; Table 1). Thus, the region responsible for substrate specificity appears to be located between the “LIPV” and “PVYG” regions (amino acid residues position: Δ4, 275-316; Δ5/Δ6, 273-314) of these enzymes, as shown in FIG. 3A.

(29) To confirm that the residues between “LIPV” to “PVYG” alone were responsible for substrate specificity, a pair of chimeric genes (Sig-7C; SEQ ID NO: 12; FIG. 5 left column, and Sig-8C; SEQ ID NO: 4; FIG. 5 right column) substituting only this region were constructed by PCR.

(30) The gene containing the Δ5/Δ6 desaturase sequence in this area and the Δ4 desaturase sequence throughout the rest of the construct desaturated only ALA (11.5±1.2%) while the reverse construct had activity only with DPA (3.8±0.2%), demonstrating that the region between amino acids “LIPV” and “PVYG” controls substrate specificity. This area includes part of the 3.sup.rd and 4.sup.th predicted transmembrane domains along with the linker region (FIG. 2).

(31) To further dissect this region, chimeric genes were constructed that divided the region between “LIPV” and “PVYG” of the S. canaliculatus Δ4 and Δ5/Δ6 desaturase into 3 fractions (end of linker to 4.sup.th transmembrane domain: “WAMT/WCLS” to “PVYG”, linker region between 3.sup.rd and 4.sup.th transmembrane domain: “TMI/IMI” to “WAMT/WCLS” and 3.sup.rd transmembrane domain to start of linker region: “LIPVF” to “TMI/IMI”).

(32) When expressed in yeast cells, Sig-9C, which is an S. canaliculatus Δ4 desaturase gene with the region encoding the amino acids from “WCLS” to “PVYG” substituted with the corresponding Δ5/Δ6 desaturase sequence (FIG. 5 left column), converted 4.0±0.5% of DPA.sub.ω3 to DHA.sub.ω3, while the reverse construct (Sig-10C; FIG. 5 right column) converted ALA to SDA at a level of 14.9±1.4%, indicating that the 4th transmembrane domain is not relevant in substrate specificity but has an effect on the catalytic activities of the enzymes.

(33) Moreover, Sig-11C (FIG. 5 left column) and its reverse gene construct (Sig-12C; FIG. 5 right column) which have sequence exchanges between the linker region of 3.sup.rd and 4th transmembrane domains (sequence from “TMI/IMI” to “WAMT/WCLS”), also retained their original substrate specificity although enzyme activity was reduced to about half of the respective wild-type genes (FIG. 5). These data suggest that the linker region between 3.sup.rd and 4th transmembrane domain (“TMI/IMI” to “WAMT/WCLS”) on the S. canaliculatus Δ4 and Δ5/Δ6 desaturases are not the critical regions for substrate specificity although sequence switching in these regions greatly reduces catalytic activities.

(34) However, when the region between the 3.sup.rd transmembrane domain and the start of linker region involving the sequence from “LIPV” to “IMI/TMI” (Sig-13C; SEQ ID NO: 14; FIG. 5 left column, and Sig-14C; SEQ ID NO: 6; FIG. 5 right column) were substituted, there was a shift in substrate specificity. The mutated S. canaliculatus Δ4 (Sig-13C; SEQ ID NO: 14; FIG. 5 left column) desaturates only ALA (13.1±1.8%) but no longer desaturates DPA.sub.ω3. Therefore this mutation allowed the gene to gain Δ5/Δ6 activity but completely abolished the Δ4 activity. In contrast, the reverse construct (Sig-14C; SEQ ID NO: 6; FIG. 5 right column) gained Δ4 activity as demonstrated by DPA.sub.ω3 desaturation (3.4±0.2%) but completely lost its original Δ5/Δ6 activity. Thus, a region that appears to be within the 3.sup.rd transmembrane domain critically affects substrate specificity.

(35) To examine whether the eight amino acids (S. canaliculatus Δ4 desaturase: LIPVFYNY-NIMMTMI; S. canaliculatusΔ5/Δ6 desaturase: LIPVFFHYQLLKIMI) between the “LIPVF” to “TMI/IMI” region were all required for substrate specificity or if only specific amino acids were critical, the inventors made constructs with alterations in this region by dividing the 8 amino acid region into two portions. Altering the “IMMT” amino acid sequence of the S. canaliculatus Δ4 desaturase gene to “LLKI” (Sig-15C; FIG. 6 left column) did not alter the substrate specificity of the Δ4 desaturase gene, as it desaturated DPA.sub.ω3(2.9±0.3%) to generate DHA.sub.ω3 and had no activity on ALA. Similarly, the reverse gene construct with alteration of the S. canaliculatus Δ5/Δ6 desaturase (Sig-16C; FIG. 6 right column) from “LLKI” to “IMMT” (corresponding residues in S. canaliculatus Δ4 desaturase) did not affect substrate specificity (16.3±1.1%, ALA). However, substitution of the Δ4 desaturase amino acid sequence “YNYN” to the Δ5/Δ6 desaturase sequence “FHYQ” at the same location (Sig-17C; SEQ ID NO: 16; FIG. 6 left column; Table 1) changed the substrate specificity from DPA.sub.ω3 to ALA (12.5±0.8%). Likewise, altering the S. canaliculatus Δ5/Δ6 desaturase sequence from “FHYQ” to the corresponding “YNYN” sequence (Sig-18C; SEQ ID NO: 8; FIG. 6 right column; Table 1) resulted in a loss of Δ5/Δ6 desaturase activity but a gain in Δ4 desaturase activity (0.5±0.1%, DPA.sub.ω3). Thus, the four amino acids that regulate the substrate specificity of S. canaliculatus Δ4 and Δ5/Δ6 desaturases are “YNYN” and the corresponding sequence “FHYQ”.

(36) TABLE-US-00001 TABLE 1 Fatty acid desaturation by yeast expressing S. canaliculatus Δ 4-desaturase, Δ5/6-desaturase and chimera constructs. Desaturation of substrates DPA DHA Conversion ALA SDA Conversion (mol %) (mol %) % (mol %) (mol %) % Δ4 5.20 ± 0.72 ± 12.12 12.01 ± 0 0 Desaturase 0.34 0.01  2.44 Δ5/6 6.88 ± 0 0  9.88 ± 2.94 ± 22.96 Desaturase 0.95  1.13 0.66 Sig-3C 7.81 ± 0.72 ± 8.48 14.49 ± 0 0 0.62 0.08  3.15 Sig-4C 8.07 ± 0 0 13.11 ± 1.87 ± 12.48 1.30  0.77 0.31 Sig-5C 8.45 ± 0 0 12.59 ± 3.81 ± 23.24 1.45  1.26 0.34 Sig-6C 6.97 ± 0.41 ± 5.49 12.19 ± 0 0 1.47 0.06  2.65 Sig-17C 6.56 ± 0 0  9.53 ± 1.37 ± 12.55 1.07  0.65 0.06 Sig-18C 7.97 ± 0.04 ± 0.50 11.36 ± 0 0 1.37 0.00  1.59
2.3 Substitution of Amino Acids within the Four Amino Acid Region Produces Enzymes with Both Δ4- and Δ6-Desaturase Activities

(37) After determining that this four amino acid region was responsible for substrate specificity, substitution of individual amino acids was performed (FIG. 7). An amino acid substitution at position 4 (FIG. 7) of the critical region showed no effect on substrate specificity in either the Δ4 (Sig-19C; FIG. 6 left column) or Δ5/Δ6 desaturases (Sig-20C; FIG. 6 right column). However, decreases in substrate conversion levels (10.0±0.4%, DPA.sub.ω3 for Sig-19C and 15.6±0.9%, ALA for Sig-20C) were observed. When a single amino acid was substituted at position 2 (N to H or vice versa), the substrate specificity of the point-mutated Δ4 desaturase (Sig-21C; FIG. 6 left column) remained the same (DPA.sub.ω3) although enzyme activity was reduced to 3.5±0.2%, which is about a 71% reduction in activity compared to wild-type. The Δ5/Δ6 desaturases with an amino acid substitution at position 2 in the critical region (Sig-22C; SEQ ID NO: 22; FIG. 6 right column) not only desaturated ALA (13.6±5.0%) but also produced DHA.sub.ω3 at a low level (0.5±0.04%). The Sig-23C (Δ4 desaturase; SEQ ID NO: 18; FIG. 6 left column) and Sig-24C (Δ5/Δ6 desaturase; SEQ ID NO: 24; FIG. 6 right column) with amino acid substitutions in the corresponding residues at position 1 of the critical region were able to desaturate both DPA.sub.ω3 and ALA. Since positions 1 and 2 seem to play a more important role in the substrate specificity, the inventors rationally engineered desaturases with two amino acid substitutions in this region (Sig-25C; SEQ ID NO: 20; FIG. 6 left column, and Sig-26C SEQ ID NO: 26; FIG. 6 right column). Interestingly, site-directed mutation of both of these amino acids concurrently generates enzymes with desaturation of DPA.sub.ω3 and ALA (Sig-25C; SEQ ID NO: 20; FIG. 6 left column, and Sig-26C SEQ ID NO: 26; FIG. 6 right column). Further, mutations including the amino acids at position 1, 2 and 4 (Sig-17C; SEQ ID NO: 16; FIG. 6 left column, and Sig-18C; SEQ ID NO: 8; FIG. 6 right column) restored specificity, even though single mutations at the 4.sup.th position did not directly affect substrate specificity as demonstrated by yeast expression (Sig-19C; FIG. 6 left column, and Sig-20C; FIG. 6 right column). This suggests that some type of interaction between these three amino acids is required for the substrate specificity of the enzymes studied here.

(38) 2.4 Four Amino Acid Residues Determine the Substrate Chain-Length and Regioselectivity of Siganus canaliculatus Δ4 and Δ5/6 Desaturases

(39) The unusually high similarity in amino acid composition (83%) between S. canaliculatus Δ4 and Δ5/Δ6 desaturases gave the present inventors an advantage in locating individual regions or residues involved in substrate specificity and regioselectivity. They identified a discrete protein region containing just four amino acid residues (“YNYN”, position 280-283 on S. canaliculatus Δ4 desaturase and the corresponding amino acids “FHYQ” at position 278-281 on the S. canaliculatus Δ5/Δ6 desaturase) which is responsible for both the substrate chain-length specificity and the regioselectivity of S. canaliculatus Δ4 and Δ5/Δ6 desaturases (FIG. 6). The absence of a crystal structure for any membrane bound desaturase made it difficult to structurally interpret the alteration in regioselectivity and substrate specificity observed here. However, the S. canaliculatus Δ4 and Δ5/Δ6 desaturases are membrane bound enzymes and, based on previous studies the histidine boxes, N- and C-termini of the enzymes have been predicted to be located on the cytoplasmic side of the membrane. Given these constraints, the membrane protein topology prediction software (TOPCONS; Bernsel et al., 2009) predicts each of these two S. canaliculatus desaturases have four transmembrane domains (amino acid residues 132-152, 159-179, 265-285, 303-323 for Δ4 desaturase and corresponding residues for Δ5/Δ6 desaturase) linked by two exoplasmic regions consisting of six and seventeen amino acids (FIG. 1). It is expected that these highly similar desaturases would share a common membrane topology and presumably a common structural folding.

(40) The four critical amino acid residues influencing the substrate specificity and regioselectivity of the S. canaliculatus desaturases are predicted to be located in the third putative transmembrane domain (FIG. 2). This four amino acid residue block is at some distance from the histidine-rich active site, which suggests that no direct interaction between the mutated amino acid residues and the substrate is required for the observed alterations in regiospecificity and substrate specificity (Cahoon et al., 1997). However, these four amino acid residues may form part of a hydrophobic substrate binding pocket which places constraints on the chain length of the substrate. Influences due to modification of the hydrophobic substrate binding pocket were observed in lipoxygenases and soluble fatty acid desaturases (Cahoon et al., 1997; Borngraber et al., 1999) and the authors suggest that the size and geometry of this hydrophobic binding pocket are responsible for the substrate specificity and insertion of double bond position. The amino acid residues “YNYN” in the critical region of the S. canaliculatus Δ4 desaturase are somewhat less bulky than the respective “FHYQ” residues in S. canaliculatus Δ5/Δ6 desaturase, possibly allowing the hydrophobic substrate binding pocket to accommodate a larger substrate such as DPA.sub.ω3 (22:5.sub.ω3), whereas the presence of bulkier residues may put a greater constraint on the substrate binding pocket to favour the smaller substrate ALA (18:2). Alternatively, the presence of a histidine residue in proximity to aromatic amino acids may result in π-π stacking interactions between aromatic amino acids which could influence packing effects and thus affect the substrate selection (Liao et al., 2013).

(41) The inventors have also generated novel desaturases with Δ4 and Δ6 activities (Sig-22C/SEQ ID NO: 22; Sig-23C/SEQ ID NO: 18; Sig-24C/SEQ ID NO: 24; Sig-25C/SEQ ID NO: 20; Sig-26C/SEQ ID NO: 26; FIG. 6) by altering only one or two amino acids at positions 1 and 2 of the critical region (FIG. 7). The dual enzyme activities of the mutants were unexpected, and mutating both residues increases enzyme activity by 2.4 to 3.3 fold compared to mutating only the amino acid residue at position 1. A single amino acid residue mutation at position 4 (FIG. 7), results in a lower catalytic ability in both Δ4 and Δ5/Δ6 desaturases but does not affect the substrate or double bond position specificities. However, when this mutation was combined with double amino acid mutations at positions 1 and 2 (FIG. 7), the Δ4 desaturase was converted to an enzyme with Δ5/Δ6 desaturase activity and substrate specificity, and vice versa. This indicates that the substrate specificity and regioselectivity of the S. canaliculatus desaturases cannot be confined to a single amino acid residue. In fact, some degree of interaction between all four amino acid residues (Δ4: “YNYN” and Δ5/Δ6: “FHYQ”) appears to be responsible for the substrate specificity and regioselectivity of the enzymes. Interestingly, similar results were obtained by Yuan et al. (1995). A single amino acid change (T231 K) in a thioesterase by itself did not affect substrate specificity but when this mutation was added to an enzyme with two mutations (M197R/R199H) which possessed equal preference for 12:0 and 14:0, the triple mutant was active only on 14:0. Here, the single mutation at position 4 (FIG. 7) shows a non-additive combinatory effect on the double mutant at positions 1 and 2, which suggests that some type of interaction is occurring between these amino acids (Sandberg and Terwillinger, 1993; Yuan et al., 1995).

Example 3

(42) Based on the results obtained in Example 2 and further sequence alignments and topology predictions, the corresponding amino acid regions controlling the substrate specificity could be identified, in Δ4, Δ5 and/or Δ6 desaturases of other organisms as well. By exchanging the corresponding amino acid regions regulating the substrate specificity, the substrate specificity can also be switched in said Δ4, Δ5 and/or Δ6 desaturases, indicating that the teaching from the S. canaliculatus Δ4 and Δ 5/6 desaturases can be readily transferred to other “front-end” desaturases. In particular, this is exemplified for the Δ6 desaturases of Ostreococcus tauri and Pythium irregulare and the delta-5 desaturase of Thraustochytrium sp., in the following. Accession numbers and/or sequence identity numbers of Δ4, Δ5 and/or Δ6 desaturases referred to in this Example have already been indicated in the description.

(43) 3.1 Ostreococcus tauri Δ6 Desaturase

(44) The following data is based on the alignment of Δ4 desaturases (Euglena gracilis, Emiliana huxleyi, Pavlova lutheri, Pavlova salina, Siganus arctica, Siganus canaliculatus) with Δ6 desaturases (Ostreococcus lucimarinus, Ostreococcus tauri, Siganus canaliculatus).

(45) 3.1.1 In the O. tauri Δ6 desaturase, the amino acid residues (AA) “MFFL” (position 283-286) correspond to the four important amino acid residues identified in the studies presented in Example 2 (S. canaliculatus Δ4-desaturase: “YNYN” and Δ5/6-desaturase: “FHYQ”)]. The amino acid residues at position 1, 2 and 4 were shown to have a significant influence on substrate specificity in S. canaliculatus. The O. lucimarinus Δ6 desaturase has the same amino acid residues at positions 1, 2 and 4 (“FWMFFLH”) as the O. tauri Δ6-desaturase. The corresponding amino acid residues on the P. salina and P. lutheri Δ4-desaturase are “FKLLFLD”.

(46) If this important region from the O. tauri Δ6 desaturase is aligned with the P. salina and P. lutheri Δ4 desaturases, the differences in the amino acid residues are: O. tauri Δ6 desaturase, “FWMFFLH” and P. lutheri/P. salina Δ4 desaturase, “FKLLFLD”. So performing amino acid residue substitutions based on these differences is feasible (Betts and Russell, 2003).

(47) Hydrophobicity: The “W” and “F” amino acid residues in the O. tauri Δ6 desaturase are aromatic amino acids which are relatively non-polar and hydrophobic. Changing from “WMF” to “KLL” will reduce the hydrophobicity of the corresponding region. Similarly, from the study with the Siganus canaliculatus Δ4 desaturase and Δ5/6 desaturase presented in Example 2, the inventors knew that the Δ5/Δ6 desaturase contains more aromatic amino acid residues in the four amino acid region, and is thus more hydrophobic than the Δ4 desaturase.

(48) All mutations based on the above are demonstrated below:

(49) TABLE-US-00002 O. tauri Δ6 desaturase: SEQ ID NO: 71 281 FWMFFLH 287/ P. lutheri and P. salina Δ4 desaturase: SEQ ID NO: 72 256/259 FKLLFLD 262/265/ O. tauri Δ6 desaturase: SEQ ID NO: 73 278 VLLFWMFFLHPSKALK 293/ Mutation 1: SEQ ID NO: 74 278 VLLFKLLFLHPSKALK 293/ Mutation 2: SEQ ID NO: 75 278 VLLFKLLFLDPSKALK 293/ Mutation 3: SEQ ID NO: 76 278 VLLFKLFFLHPSKALK 293/ Mutation 4: SEQ ID NO: 77 278 VLLFKLFFLDPSKALK 293/ Mutation 5: SEQ ID NO: 78 278 VLLFKMLFLHPSKALK 293/ Mutation 6: SEQ ID NO: 79 278 VLLFKMLFLDPSKALK 293/ Mutation 7: SEQ ID NO: 80 278 VLLFWLFFLHPSKALK 293/ Mutation 8: SEQ ID NO: 81 278 VLLFWLFFLDPSKALK 293/ Mutation 9: SEQ ID NO: 82 278 VLLFWMLFLHPSKALK 293/ Mutation 10: SEQ ID NO: 83 278 VLLFWMLFLDPSKALK 293/ Mutation 11: SEQ ID NO: 84 278 VLLFWLLFLHPSKALK 293/ Mutation 12: SEQ ID NO: 85 278 VLLFWLLFLDPSKALK 293/ Mutation 13: SEQ ID NO: 86 278 VLLFKMFFLHPSKALK 293/ Mutation 14: SEQ ID NO: 87 278 VLLFWMFFLDPSKALK 293/

(50) 3.1.2 The 3.sup.rd transmembrane (TM) domain of S. canaliculatus Δ4 and Δ5/Δ6 desaturases contains the region of interest, as demonstrated in Example 2. A topology prediction (TOPCONS) was used to identify the 3.sup.rd transmembrane domain of the O. tauri Δ6 desaturase from positions 300-320. Alignment of this region with P. salina, S. artica or P. lutheri Δ4 desaturases identifies a region of homology which can be used to replace the O. tauri 3.sup.rd transmembrane region; i.e. the bold O. tauri Δ6 desaturase region can be replaced by the bold region of Δ4 desaturase genes which could result in a change of the substrate specificity of the Δ6 desaturase to the substrate specificity of a Δ4 desaturase. Tequence identity numbers only refer to sections in bold lettering.

(51) TABLE-US-00003 O. tauri Δ6 desaturase: SEQ ID NO: 88 290 KALKGGKYEELVWMLAAHVIRTWTIKAVTGFTAMQSYGLFL 330/ P. lutheri Δ4 desaturase: SEQ ID NO: 89 274 EKISPLARALFAPAVACKLGFWARFVALPLWLQPTVHTALCIC 316/ S. artica Δ4 desaturase: SEQ ID NO: 90 277 EKLPESYR-KERNIAIGLRVFFFIRKFVVPFALHFSWYTLLCTY 319/ P. salina Δ4 desaturase: SEQ ID NO: 91 280 SKLAGYLFMPSLLLKLTFWARFVALPLYLAPSVHTAVCIA 319/

(52) 3.1.3 The 3.sup.rd transmembrane domain of O. tauri Δ6 desaturase (position 300-320, in bold) can be replaced with the corresponding Δ4 desaturase 3.sup.rd transmembrane domain (in bold) based on TOPCONS topology prediction software.

(53) TABLE-US-00004 O. tauri Δ6 desaturase: (sequence identity numbers only refer to sections in bold lettering) SEQ ID NO: 88 290 KALKGGKYEELVWMLAAHVIRTWTIKAVTGFTAMQSYGLFL 330/ a) Substitute with P. lutheri Δ4 desaturase (281- 301): SEQ ID NO: 92 RALFAPAVACKLGFWARFVAL/ b) Substitute with P. salina Δ4 desaturase (291- 311): SEQ ID NO: 93 LLLKLTFWARFVALPLYLAPS/ c) Substitute with S. arctica Δ4 desaturase (293- 313): SEQ ID NO: 94 LRVFFFIRKFVVPFALHFSWY/

(54) 3.1.4 Substitute the entire corresponding “LI PV” to “PVYG” region on O. tauri Δ6 desaturase (position 278-321) with the respective Δ4 desaturase domain, based on sequences alignments. This region was shown to be important in the S. canaliculatus desaturase sequences, as shown in Example 2.

(55) TABLE-US-00005 O. tauri Δ6 desaturase (position 278-321): SEQ ID NO: 95 VLLFWMFFLHPSKALKGGKYEELVWMLAAHVIRTWTIKAVTGFT/

(56) Substitute with:

(57) TABLE-US-00006 a) P. lutheri Δ4 desaturase (265-307): SEQ ID NO: 96 ELLAWRWEGEKISPLARALFAPAVACKLGFWARFVALPLWLQP/ b) P. salina Δ4 desaturase (268-310): SEQ ID NO: 97 ELVMWRWEGEPISKLAGYLFMPSLLLKLTFWARFVALPLYLAP/ c) S. artica Δ4 desaturase (268-310): SEQ ID NO: 98 DLIEMKYKGEKLPESYRKERNIAIGLRVFFFIRKFVVPFALHF/

(58) 3.1.5 The four amino acid region of the S. canaliculatus Δ4 and Δ5/Δ6 desaturases identified in Example 2 are located at the end of 3.sup.rd transmembrane domain close to the exoplasmic face of the membrane (positions 280-283 and 278-281, respectively). Thus, based on topology predictions, the comparable amino acid residues on the 3.sup.rd transmembrane domain of the O. tauri Δ6 desaturase are “KAVT” (position 315-318). Substituting said amino acid residues with the Δ4 desaturase amino acid residues from the same topological location can influence the substrate specificity and regioselectivity.

(59) O. tauri (position 315-318) “KAVT”/SEQ ID NO: 99 replace with:

(60) P. lutheri Δ4 desaturase (position 296-299): “ARFV”/SEQ ID NO: 100

(61) or P. salina Δ4 desaturase (position 306-309): “LYLA”/SEQ ID NO: 101

(62) or S. artica Δ4 desaturase (position 308-311): “LHFS”/SEQ ID NO: 102

(63) 3.2 Thraustochytrium sp. Δ5 Desaturase

(64) The following data is based on the sequence alignment of the Δ4 desaturase (Pavlova lutheri, Pavlova saline, Siganus artica, Thraustochytrium sp., Siganus canaliculatus) with the Δ5 desaturase (Thraustochytrium sp., Pavlova saline, Siganus canaliculatus).

(65) 3.2.1 In the Thraustochytrium sp. Δ5 desaturase, the amino acid residues corresponding to the region identified in S. canaliculatus are “TLYL” (position 270-273), with positions 2, 3, and 4 conserved with the P. saline Δ5 desaturase. For conversion to a Δ4 desaturase, substitute with the bold region (sequence identity numbers only refer to sections in bold lettering):

(66) TABLE-US-00007 Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 103 265 IGLGW HPR 276/ P. lutheri Δ4 desaturase: SEQ ID NO: 104 265 ELLAW EKI 276/ P. salina Δ4 desaturase: SEQ ID NO: 105 268 ELVMW EPI 279/ S. artica Δ4 desaturase: SEQ ID NO: 106 268 DLIEM EKL 279/
a) Substitute one amino acid residue at a time. Bold: substituted amino acid residue/SEQ ID NO.

(67) Replace with “RWEG” (corresponding region on P. saline and P. lutheri Δ4 desaturase)

(68) TABLE-US-00008 Mutation 1: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 107 265 IGLGWRLYLHPR 276/ Mutation 2: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 108 265 IGLGWRWYLHPR 276/ Mutation 3: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 109 265 IGLGWRWELHPR 276/ Mutation 4: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 110 265 IGLGWRWYGHPR 276/ Mutation 5: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 111 265 IGLGWRLYGHPR 276/ Mutation 6: Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 112 265 IGLGWRLELHPR 276/
b) Replace with “KYKG” (corresponding region on S. artica Δ4 desaturase). Bold: substituted amino acid residue/SEQ ID NO.

(69) TABLE-US-00009 Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 113 265 IGLGWTLYLHPR 276/ Mutation 1: SEQ ID NO: 114 265 IGLGWKLYLHPR 276/ Mutation 2: SEQ ID NO: 115 265 IGLGWKYYLHPR 276/ Mutation 3: SEQ ID NO: 116 265 IGLGWKYKLHPR 276/ Mutation 4: SEQ ID NO: 117 265 IGLGWKLKLHPR 276/ Mutation 5: SEQ ID NO: 118 265 IGLGWKLYGHPR 276/ Mutation 6: SEQ ID NO: 119 265 IGLGWKLKGHPR 276/ Mutation 7: SEQ ID NO: 120 265 IGLGWTYYLHPR 276/ Mutation 8: SEQ ID NO: 121 265 IGLGWTYKLHPR 276/ Mutation 9: SEQ ID NO: 122 265 IGLGWTYKGHPR 276/ Mutation 10: SEQ ID NO: 123 265 IGLGWTLKLHPR 276/ Mutation 11: SEQ ID NO: 124 265 IGLGWTLKGHPR 276/

(70) 3.2.2 Based on sequence alignments, there are five amino acid residues near the important region that are conserved between P. lutheri and P. saline Δ4 desaturases. Replacing these 5 amino acid residues can result in a change in desaturase specificity (sequence identity numbers only refer to sections in bold lettering):

(71) TABLE-US-00010 Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 125 265 IGLGWTLYLHPR 276/ P. lutheri 44 desaturase: SEQ ID NO: 126 265 ELLAWRWEGEKI 276/ P. salina 44 desaturase: SEQ ID NO: 127 268 ELVMWRWEGEPI 279/ After substitution, Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 128 265 IGLGWRWEGEPR 276/

(72) 3.2.3 Based on sequence alignments, the region corresponding from “LIPV” to “PVYG” identifled in Example 2 is from position 265-307, in the amino acid sequence of the Thraustochytrium sp. Δ5 desaturase. Substitution of amino acid residues from the P. lutheri Δ4 desaturase is shown below.

(73) TABLE-US-00011 Thraustochytrium sp. Δ5 desaturase: SEQ ID NO: 129 265 IGLGWTLYLHPRYMLRTKRHMEFVWIFARYIGWFSLMGALGYS 307/ P. lutheri Δ4 desaturase: SEQ ID NO: 130 265 ELLAWRWEGEKISPLARALFAPAVACKLGFWARFVALPLWLQP 307/

(74) 3.2.4 Based on protein topology prediction software (TOPCONS), the Thraustochytrium sp. Δ5 desaturase has four transmembrane domains and two hydrophobic stretches. The 3.sup.rd transmembrane domain of Thraustochytrium sp. Δ5 desaturase is located from position 287-307. Substitution of the 3.sup.rd transmembrane domain with the corresponding domain based on homology is shown below:

(75) TABLE-US-00012 Thraustochytrium sp. Δ5 desaturase, 3.sup.rd transmembrane domain: SEQ ID NO: 131 287 FVWIFARYIGWFSLMGALGYS 307/

(76) Replace with:

(77) TABLE-US-00013 P. lutheri Δ4 desaturase: SEQ ID NO: 132 287 AVACKLGFWARFVALPLWLQP 307/ or P. salina Δ4 desaturase: SEQ ID NO: 133 290 SLLLKLTFWARFVALPLYLAP 310/ or S. artica Δ4 desaturase: SEQ ID NO: 134 290 AIGLRVFFFIRKFVVPFALHF 310/

(78) Based strictly on topology predictions (TOPCONS), the inventors could substitute the 3rd transmembrane domain of the Thraustochytrium sp. Δ5 desaturase with the 3rd transmembrane domain of other Δ4 desaturases:

(79) TABLE-US-00014 Thraustochytrium sp. Δ5 desaturase, 3.sup.rd transmembrane domain: SEQ ID NO: 131 287 FVWIFARYIGWFSLMGALGYS 307/

(80) Replace with:

(81) TABLE-US-00015 P. lutheri Δ4 desaturase, 3rd TM: SEQ ID NO: 135 281 RALFAPAVACKLGFWARFVAL 301/ or P. Salina Δ4 desaturase, 3rd TM: SEQ ID NO: 136 291 LLLKLTFWARFVALPLYLAPS 311/ or S. arctica Δ4 desaturase, 3rd TM: SEQ ID NO: 137 293 LRVFFFIRKFVVPFALHFSWY 313/

(82) 3.2.5 Based on the studies shown in Example 2, the four amino acid residues which influence the substrate specificity are located at the end of the 3rd transmembrane domain close to the exoplasmic face. The four amino acid residues are located in the last 6 amino acid residues in the 3.sup.rd transmembrane domain of S. canaliculatusΔ4 and Δ5/Δ6 desaturases. In Thraustochytrium sp. Δ5 desaturase, position 302-305 represents the same location based on topology prediction (starts at last 6 amino acid residues in the 3rd transmembrane domain).

(83) a) Substitution of amino acid residues corresponding to Thraustochytrium sp. Δ5 desaturase position 302-305 (“GALG”/SEQ ID NO: 138) based on sequence alignment as follows:

(84) TABLE-US-00016 i) P. lutheri Δ4 desaturase: SEQ ID NO: 139 302-305 ″PLWL″/ ii) P. salina Δ4 desaturase: SEQ ID NO: 140 305-308 ″PLYL″/ iii) S. artica Δ4 desaturase: SEQ ID NO: 141 305-308 ″PFAL″/
b) Substitution of amino acid residues corresponding to Thraustochytrium sp. Δ5 desaturase 302-305 (“GALG”/SEQ ID NO: 138) based on topology prediction, as follows:

(85) TABLE-US-00017 i) P. lutheri Δ4 desaturase: SEQ ID NO: 142 296-299 ″ARFV″/ ii) P. salina Δ4 desaturase: SEQ ID NO: 143 306-309 ″LYLA″/ iii) S. artica Δ4 desaturase: SEQ ID NO: 144 308-311 ″LHFS″/
3.3 Pythium irregulare Δ6 Desaturase

(86) The following data is based on sequence alignments of the Pythium irregulare Δ6 desaturase with other Δ6 desaturases (Siganus canaliculatus.) Primula farinosa) and Δ4 desaturases (Emiliana huxleyi, Monosiga brevicollis, Pavlova lutheri, Pavlova salina, Siganus artica, Thalassiosira pseudonana, Siganus canaliculatus, Thraustochytrium sp.).

(87) 3.3.1 The amino acid residues corresponding to the region identified in S. canaliculatus in Example 2, in the P. irregulare Δ6 desaturase are “AQSF” (position 284-287). These residues are conserved between several Δ6 desaturases (P. irregulare, P. farinosa). Based on sequence alignment, these amino acid residues could be substituted with amino acid residues from Δ4 desaturases (sequence identity numbers only refer to sections in bold lettering):

(88) TABLE-US-00018 P. irregulare Δ6 desaturase: SEQ ID NO: 145 280 LSWLAQSFFYV 290/

(89) Replace with amino acid residues from:

(90) TABLE-US-00019 P. salina Δ4 desaturase: SEQ ID NO: 146 259 FKLLFLDISEL 269/ P. lutheri Δ4 desaturase: SEQ ID NO: 147 256 FKLLFLDALEL 266/ S. artica Δ4 desaturase: SEQ ID NO: 148 259 FQWVFLGLHDL 269/  M. brevicollis Δ4 desaturase: SEQ ID NO: 149 241 AKVIIGDWYNL 251/ E. huxleyi Δ4 desaturase: SEQ ID NO: 150 234 FVFAFTIRKYA 244/ T. pseudonana Δ4 desaturase: SEQ ID NO: 151 315 LAKVFQQDFEV 325/ Thraustochytrium sp. Δ4 desaturase: SEQ ID NO: 152 290 INKVVTQDVGV 300/

(91) Substitution of single amino acid residues could also be performed as described above.

(92) 3.3.2 Based on topology prediction (TOPCONS), the P. irregulare Δ6 desaturase contains four transmembrane domains and two hydrophobic stretches; the 3rd transmembrane domain is located between position 310-330. From Example 2 it is known, that the amino acid residues of interest are in the third transmembrane domain. Based on sequence alignment, the inventors could substitute the 3rd transmembrane domain of the P. irregulare Δ6 desaturase with the corresponding region from Δ4 desaturases.

(93) TABLE-US-00020 3rd TM P. irregulare Δ6 desaturase: SEQ ID NO: 153 310 AGLIVHYIWQLAIPYFCNMSL 330/

(94) Replace with:

(95) TABLE-US-00021 P. lutheri Δ4 desaturase: SEQ ID NO: 154 288 VACKLGFWARFVALPLWLQPT 308/ or P. salina Δ4 desaturase: SEQ ID NO: 155 291 LLLKLTFWARFVALPLYLAPS 311/ or S. artica Δ4 desaturase: SEQ ID NO: 156 291 IGLRVFFFIRKFVVPFALHFS 311/ or Thraustochytrium sp. Δ4 desaturase: SEQ ID NO: 157 325 WIMKALTVLYMVALPCYMQGP 345/ or M. brevicollis Δ4 desaturase: SEQ ID NO: 158 274 VLARICWLVRLVAIPVYLHGW 294/

(96) 3.3.3 Substitute the 3rd transmembrane domain of the P. irregulare Δ6 desaturase with the 3rd transmembrane domain of other Δ4 desaturase based on topology predictions:

(97) TABLE-US-00022 3rd TM P. irregulare Δ6 desaturase: (SEQ ID NO: 153) 310 AGLIVHYIWQLAIPYFCNMSL 330/

(98) Replace with:

(99) TABLE-US-00023 P. lutheri Δ4 desaturase, 3rd TM: SEQ ID NO: 159 281 RALFAPAVACKLGFWARFVAL 301/ or P. salina Δ4 desaturase, 3rd TM: SEQ ID NO: 160 291 LLLKLTFWARFVALPLYLAPS 311/ or S. arctica Δ4 desaturase, 3rd TM: SEQ ID NO: 161 293 LRVFFFIRKFVVPFALHFSWY 313/

(100) 3.3.4 Based on the studies shown in Example 2, the four amino acid residues influencing the substrate specificity are located at the end of 3.sup.rd transmembrane domain close to the exoplasmic face of the membrane. In the P. irregulare Δ6 desaturase, position 325-328 (“FCNM”) represents the same location, based on topology predictions. This region can be replaced as outlined below:

(101) a) Substitute with amino acid residues corresponding to P. irregulare Δ6 desaturase position 325-328 (“FCNM”/SEQ ID NO: 162), based on sequence alignment:

(102) TABLE-US-00024 i) P. lutheri Δ4 desaturase: SEQ ID NO: 163 303-306 ″LWLQ″/ ii) P. salina Δ4 desaturase: SEQ ID NO: 164 306-309 ″LYLA″/ iii) S. artica Δ4 desaturase: SEQ ID NO: 165 306-309 ″FALH″/ iv) Thraustochytrium sp. Δ4 desaturase: SEQ ID NO: 166 340-343 ″CYMQ″/
b) Substitute with amino acid residues corresponding to P. irregulare Δ6 desaturase position 325-328 (“FCNM”/SEQ ID NO: 162), based on topology prediction:

(103) TABLE-US-00025 i) P. lutheri Δ4 desaturase: SEQ ID NO: 167 296-299 ″ARFV″/ ii) P. salina Δ4 desaturase: SEQ ID NO: 168 306-309 ″LYLA″/ iii) S. artica Δ4 desaturase: SEQ ID NO: 169 308-311 ″LHFS″/ iv) Thraustochytrium sp. Δ4 desaturase: SEQ ID NO: 170 337-340 ″ALPC″/ 

REFERENCES

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