The pathway, also known as PUFA synthase pathway or the de novo synthesis, involves a single enzyme complex from the polyketide synthase (PKS) family — PUFA synthase. The synthase takes up metabolite acetyl-CoA as the starter unit, and takes up malonyl-CoA as the building blocks. It works iteratively to lengthen and reduce the fatty acid chain without releasing the acyl chain from the complex, until the synthesis of DHA finishes after 10 cycles. PUFA synthase enzymes catalyzing the synthesis of DHA are found both in eukaryotic microalgae and prokaryotic myxobacteria, while the exact mechanism of the pathway is still not well-known and under intensive study.

By comparing data from several existing research, we choose to introduce a eukaryotic polyketide synthase pathway originally found in microalgae Schizochytrium sp., which has a relatively high output in comparison to its prokaryotic counterpart, and requires less resources (i.e., NADPH, oxygen) than the aerobic DHA synthesis pathway.

Graph 1. Predicted structure of PUFA synthase subunits with Alphafold 3. From left to right: Subunit A, B and C.

Graph 2. A graph showing the chemical reactions and role of different domains. This one is from paper, to be replaced.

The DHA-synthesis machine — PUFA synthase consists of three subunits and requires a transferase enzyme for its activation. Each subunit contains multiple functional domains that play different roles during each iterative cycle to synthesize DHA:

• Enoyl-reductase (ER)
• Acyl-carrier protein (ACP)
• Dehydratase (DH)
• Keto-reductase (KR)
• Keto-synthase (KS)
• MAT
• Acyl-transferase (AT)

To introduce the enzyme into Y. lipolytica, we put each of the subunits under control of a common constitutive promoter TEF1. It is reported that the RBS region is contained in the promoter sequence. Each subunit is His-tagged in order to detect their expression. After each coding sequence, a common LIP2 terminator and a spacer is added.

We plan to combine our central pathway with various optimization strategies to increase the ultimate yield, as the same time balancing growth and production, which include:

• Knocking out genes KU70 and PEX10 by homologous recombination to efficiently prevent PUFA degradation.

PEX10 encodes a key protein for peroxisomal beta-oxidation in Y. lipolytica [15]. Inspired by https://2022.igem.wiki/nnu-china/ , to prevent the degradation of synthesized DHA through beta-oxidation, we plan to knock out the PEX10 gene in Y. lipolytica by homologous recombination (HR). KU70, a key gene enabling non-homologous end-joining in Y. lipolytica, may first need to be knocked out to increase the chance of HR, which is originally low in Y. lipolytica.

The knockout is carried out by replacing part of the PEX10 gene with the LEU2 gene construct, which is used for nutrient auxotrophy selection. The construct is flanked with 2 loxP sites, so that after the knockout, the LEU2 marker gene can be released using the Cre-loxP system by introducing another plasmid containing the Cre recombinase and a hygromycin resistance gene for selection, enabling next round of transformation and selection with the same marker.

• Replenishing reactant NADPH supply through metabolic engineering. At least 14 NADPH molecules are consumed to provide the redox power for generating a DHA molecule. Various literatures have reported NADPH being a limiting factor in lipid production[13-14]. We plan to express the GapC gene encoding a NADP+-dependent G3P dehydrogenase in Y. lipolytica, which converts NADH to NADPH, increasing its availability during DHA synthesis.

In the project, we not only consider metabolic strategies to increase the yield of DHA. Currently DHA production requires harvesting and lysis of the cell, which is unsustainable. However, DHA is a large molecule that would be difficult to transport out through the cell membrane efficiently. To address this issue, we will also explore possible strategies for DHA secretion through our modeling, aiming to achieve a continuous production.