Difference between revisions of "Part:BBa K4343073"

Revision as of 14:44, 9 October 2022

pUC-HUH-FmDes12

FmDes12 gene encodes a ∆-12 desaturase from Fusarium moniliform, which can catalyze C18:1 to C18:2. This gene is expressed in Y. lipolytica.

Characterisation

We constructed a plasmid containing promoter TEF, FmDes12, terminator lip2t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into Yarrowia lipolytica. The segment was 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. It was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids it produced was 7.3%. Similarly, Po1f-18 was obtained by integrating two copies of FmDes12 and the proportion of EPA was 9.6% (Fig: Po1f-4).

Sequence and Features