Part:BBa_K4343069
pUC-HUH-PaDes17(LEU)
The PaDes17 gene encodes a ∆-17 elongase from Pythium aphanidermatum that catalyzes the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA), an ω 3 polyunsaturated fatty acid (PUFA).
Characterisation
We constructed a plasmid containing promoter TEF, PaDes17, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered Yarrowia lipolytica and randomly integrated into its genome. The engineered strain Po1f-2 was obtained. The PaDes17 gene was expressed in the presence of the promoter TEF, which can catalyze the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA). The activity of PaDes17 was examined by gas chromatography.
Result
We integrate one copy of codon-optimized PaDes17 gene from Euglena gracilis under the control of strong promoter PTEF into the genome of engineered strain with one copy of EgDes5 to obtain the engineered strain Po1f-1 by non-homologous end joining (NHEJ) Fig(po1f-2) showed that the engineered strain Po1f-2 was able to produce 16.3% of arachidonic acid (ARA; C20:4 n-6) and 2.4% of EPA in the TFAs, which enabled the synthesis of EPA in the Po1f.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2102
Illegal XhoI site found at 667
Illegal XhoI site found at 696
Illegal XhoI site found at 1844 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1239
Illegal AgeI site found at 1667
Illegal AgeI site found at 2769 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI site found at 1242
Illegal BsaI site found at 1670
Illegal BsaI site found at 2772
Illegal BsaI.rc site found at 1236
Illegal BsaI.rc site found at 1664
Illegal BsaI.rc site found at 2766
Illegal BsaI.rc site found at 3036
Illegal SapI site found at 1078
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