Part:BBa_K4343068

pUC-HUH-EgDes5

The EgDes5 gene encodes a ∆-5 desaturase from Euglena gracilis that catalyzes the conversion of dihomo-γ-linolenic acid (DGLA; C20: 3n-6) to arachidonic acid (ARA; C20:4 n-6).

Characterisation

We constructed a plasmid containing promoter TEF, EgDes5, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered Yarrowia lipolytica Po1f-1 and randomly integrated into its genome. EgDes5 gene was expressed under control of promoter TEF, which could catalyze the formation of arachidonic acid (ARA; C20:4 n-6) from eicosadienoic acid (EDA; C20:2n-6). The activity of EgDes5 was examined by gas chromatography.

Result

We integrate one copy of codon-optimized EgDes5 gene from Euglena gracilis under the control of strong promoter PTEF into the genome to obtain the engineered strain Po1f-1 by non-homologous End Joining (NHEJ) The percentage of arachidonic acid (ARA; C20:4 n-6) were 19.4%.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1920
  Illegal XhoI site found at 1953
  Illegal XhoI site found at 4708
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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 5629
  Illegal AgeI site found at 6036
  
• 1000
  INCOMPATIBLE 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 6039
  Illegal BsaI.rc site found at 4661
  Illegal BsaI.rc site found at 6033
  Illegal BsaI.rc site found at 6303
  Illegal SapI.rc site found at 363
  Illegal SapI.rc site found at 3427