Part:BBa_K5159001

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 (BBa_K5159002) and subunit C (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. In ORF_A, main identified domains via 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. 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).