Part:BBa_K4343072
pUC-HUH-MaElo2
MaElo2 gene encodes a C16/18 elongase from Mortierella alpina, which can catalyze Palmitic acid (C16:0) to Stearic acid (C18:0). This gene is expressed in Y. lipolytica.
Characterisation
We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into Yarrowia lipolytica. This segment would be randomly integrated into its genome. FmDes12 gene was expressed under the control of TEF promoter. Then the activity of FmDes12 was tested by gas chromatography.
Result
In order to reduce the amount of intermediates and increase the amount of EPA, elongases/desaturases above were randomly integrated of several copies in the genome. Besides, the optimal copy numbers of the elongases/desaturases were detected by qPCR. Firstly, MaElo2 was randomly integrated into the Po1f-3 genome because the content of C16:0 accounted for about 15% of the TFAs of strain Po1f-3. And it was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids was 7.3%. (Fig: Polf-17)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1920
Illegal XhoI site found at 1953 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2108
Illegal AgeI site found at 97
Illegal AgeI site found at 458
Illegal AgeI site found at 3161
Illegal AgeI site found at 3522
Illegal AgeI site found at 4664
Illegal AgeI site found at 5514 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI site found at 638
Illegal BsaI site found at 3702
Illegal BsaI site found at 4239
Illegal BsaI site found at 4667
Illegal BsaI site found at 5517
Illegal BsaI.rc site found at 4661
Illegal BsaI.rc site found at 5511
Illegal BsaI.rc site found at 5781
Illegal SapI.rc site found at 363
Illegal SapI.rc site found at 3427
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