Coding

Part:BBa_K5159003

Designed by: Tianyue WU   Group: iGEM24_HKUST   (2024-09-23)


Schizochytrium sp. ATCC 20888 PUFA synthase ORF_C with His-tag

This gene encodes PUFA synthase subunit C, the last of the three subunits that assembles together to form the DHA-synthesizing PUFA synthase enzyme cluster in cytoplasm, together with subunit A (Part:BBa_K5159001) and subunit B (Part:BBa_K5159002). The sequence is codon-optimized for oleaginous yeast Yarrowia lipolytica and a His-tag with a linker sequence is attached to the C terminus to facilitate the enzyme expression detection.

ORF_C contains sequences corresponding to the following functional domains, with sequence annotations analyzed by InterPro:

  • Dehydratase (DHc)

There are also 2 DH domains identified on the PUFA synthase cluster. DH domain is in charge of the second step of desaturation, by catalyzing the formation of double bond while releasing one water molecule. Different DH domains can affect the position of double bond by isomerization and following steps, leading to a different final product. DHc subsequently catalyzes the 2, 2 or 2, 3-isomerization of the double bond, preparing the acyl-chain for a next elongation cycle or release (Zhang, 2022).

  • Enoyl-reductase (ERc)

According to the previous description, a double bond is formed after each elongation step, leading to 10 double bonds formed after 10 elongation cycles, which is impossible in chemical structure. For DHA synthesis, 6 double bonds are the right amount, so at certain steps the double bond formed must be further reduced, again consuming one NADPH, catalyzed by the ER domain.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2354
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 970
    Illegal XhoI site found at 100
    Illegal XhoI site found at 466
    Illegal XhoI site found at 913
    Illegal XhoI site found at 2916
    Illegal XhoI site found at 2983
    Illegal XhoI site found at 3268
    Illegal XhoI site found at 3343
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 589
    Illegal NgoMIV site found at 619
    Illegal NgoMIV site found at 1711
    Illegal NgoMIV site found at 2257
    Illegal NgoMIV site found at 3199
    Illegal NgoMIV site found at 3232
    Illegal NgoMIV site found at 3445
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2322
    Illegal BsaI site found at 3504
    Illegal BsaI site found at 4150
    Illegal BsaI.rc site found at 460
    Illegal BsaI.rc site found at 3121
    Illegal BsaI.rc site found at 4126
    Illegal BsaI.rc site found at 4462
    Illegal SapI.rc site found at 2463

Reference

[1] Guo P, Dong L, Wang F, Chen L and Zhang W (2022), Deciphering and engineering the polyunsaturated fatty acid synthase pathway from eukaryotic microorganisms. Front. Bioeng. Biotechnol. 10:1052785. https://doi.org/10.3389/fbioe.2022.1052785

[2] Schizochytrium sp. ATCC_20888 polyunsaturated fatty acid synthase subunit C gene, complete cds. GenBank: AF378329.2. https://www.ncbi.nlm.nih.gov/nuccore/AF378329


[edit]
Categories
Parameters
None