Coding

Part:BBa_K5159002

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


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

This gene encodes PUFA synthase subunit B, the second 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 C (Part:BBa_K5159003). 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_B contains sequences corresponding to the following functional domains, with sequence annotations analyzed by InterPro:

  • Ketoacyl-ACP synthase (KSB)

There are 2 Ketoacyl-ACP synthase domains identified on the PUFA synthase cluster. KS domain is mainly in charge of the elongation of the acyl chain by 2 carbon at a time, interacting with the acyl-carrier protein (ACP) domains carrying a malonyl group. One CO2 molecule is released during each elongation step.

  • Chain length factor (CLF)

As its name implies, CLF is one of the main regions that determines the product length in terms of carbon number. Its structure assembles KSB instead of missing a few key catalytic sites. It forms dimer with the KSB domain, between which provides a docking cleft for the acyl-chain elongation (Zhang, 2022).

  • Acyltransferase (AT)

AT domain is in charge of the release of the final product as free fatty acid from the enzyme complex, in our case mainly DHA. The release step is considered to be different in prokaryotic and eukaryotic PUFA synthases, where in the prokaryotic system, the product will likely be released as phospholipid by an AGPAT domain and can be further converted into free fatty acid or triacylglycerol (TAG).

  • Enoyl-reductase (ERB)

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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1762
    Illegal XhoI site found at 2008
    Illegal XhoI site found at 2323
    Illegal XhoI site found at 2914
    Illegal XhoI site found at 3286
    Illegal XhoI site found at 4404
    Illegal XhoI site found at 5032
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1117
    Illegal NgoMIV site found at 3961
    Illegal NgoMIV site found at 4861
    Illegal NgoMIV site found at 4894
    Illegal NgoMIV site found at 5434
    Illegal AgeI site found at 2488
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1273
    Illegal BsaI site found at 3463
    Illegal BsaI site found at 5166
    Illegal BsaI.rc site found at 1195
    Illegal BsaI.rc site found at 1246
    Illegal BsaI.rc site found at 2011
    Illegal BsaI.rc site found at 2929
    Illegal BsaI.rc site found at 4006
    Illegal BsaI.rc site found at 4471
    Illegal BsaI.rc site found at 4606

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 B gene, complete cds. GenBank: AF378328.2. https://www.ncbi.nlm.nih.gov/nuccore/158518690


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