Coding

Part:BBa_K5159001

Designed by: Tianyue WU   Group: iGEM24_HKUST   (2024-09-20)

Schizochytrium sp. ATCC 20888 PUFA Synthase ORF_A with His-tag

This gene encodes PUFA synthase subunit A, the first and largest of the three subunits that assembles together to form the DHA-synthesizing PUFA synthase enzyme cluster in cytoplasm, together with subunit B (Part:BBa_K5159002) 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.

There are multiple functional domains identified on each PUFA synthase subunit, with sequence annotations analyzed by InterPro. In ORF_A, main identified domains via the alignment, sequentially from 5’ end to 3’ end, are:

  • Ketoacyl-ACP synthase (KSa)

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.

  • Malonyl-CoA:ACP transacylase (MAT)

MAT domain transfers the malonyl group from malonyl-CoA substrate to ACP domains to enter the reaction. It is crucial to the initiation of the elongation steps. In contrast, the acyltransferase domain on subunit B helps release the final product from the ACP domain, corresponding to the termination step of the whole reaction.

  • Tandem repeats of Acyl-carrier protein sequence (ACP), where in our sequence 9 repeats are identified

ACP domains “hold” the acyl chain throughout the entire reaction, “rotating” it to interact with other different domains for certain reactions without releasing fatty acid byproducts from the enzyme cluster until reaching the final step, which characterizes the de novo DHA-synthesis pathway with a high product specificity. Increased copy number of ACP leads to an increased binding probability of the domain with the substrate malonyl-CoA, which can potentially accelerate DHA synthesis.

  • Ketoreductase (KR)

After elongation, the KR domain reduces one ketone group to hydroxyl group by consuming 1 NADPH from other metabolic pathways. This is the first step towards desaturation, which means introducing a double bond. Unlike other domains, the KR domain only exists on ORF_A but not on other subunits.

  • Dehydratase (DHa)

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 (Zhang, 2022).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 6719
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 206
    Illegal BglII site found at 2009
    Illegal BglII site found at 2069
    Illegal XhoI site found at 1414
    Illegal XhoI site found at 3445
    Illegal XhoI site found at 3688
    Illegal XhoI site found at 3772
    Illegal XhoI site found at 4105
    Illegal XhoI site found at 4341
    Illegal XhoI site found at 4354
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 121
    Illegal NgoMIV site found at 346
    Illegal NgoMIV site found at 946
    Illegal NgoMIV site found at 1366
    Illegal NgoMIV site found at 2083
    Illegal NgoMIV site found at 2107
    Illegal NgoMIV site found at 5581
    Illegal AgeI site found at 715
    Illegal AgeI site found at 1054
    Illegal AgeI site found at 7552
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1192
    Illegal BsaI site found at 2481
    Illegal BsaI site found at 6490
    Illegal BsaI site found at 7164
    Illegal BsaI site found at 7555
    Illegal BsaI.rc site found at 109
    Illegal BsaI.rc site found at 1561
    Illegal BsaI.rc site found at 2506
    Illegal BsaI.rc site found at 2536
    Illegal BsaI.rc site found at 2686
    Illegal BsaI.rc site found at 3415
    Illegal BsaI.rc site found at 3448
    Illegal SapI site found at 1809
    Illegal SapI site found at 2514
    Illegal SapI site found at 8082

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] Hayashi, S., Satoh, Y., Ujihara, T. et al (2016). Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins. Sci Rep 6, 35441. https://doi.org/10.1038/srep35441

[3] Schizochytrium sp. ATCC_20888 polyunsaturated fatty acid synthase subunit A gene, complete cds. GenBank: AF378327.2. https://www.ncbi.nlm.nih.gov/nuccore/AF378327


[edit]
Categories
Parameters
None