To construct Yarrowia lipolytica engineered strains rich in DHA and EPA, we need to sequentially construct the metabolic pathways of DHA and EPA. On this basis, various metabolic engineering strategies were adopted to increase the titers of DHA and EPA. Since EPA is a precursor for DHA synthesis, our experimental design was to construct a metabolic pathway for EPA synthesis with the help of homologous recombination gene editing technology. Further combined with a “push-and-pull” strategy to unblock the EPA metabolic pathway, the fatty acid β-oxidation pathway increased EPA titers. Finally, the engineered strain with high EPA production was selected and two heterologous genes were introduced to construct the DHA synthesis pathway. The DHA content of the engineered strain was determined by shaking flask fermentation.

Yarrowia lipolytica (hereafter Y. lipolytica) is described as a new type of unconventional oleaginous yeast, whose oil content exceeds 50% of its dry weight. Meanwhile, lipolytica is generally regarded as a safe strain (GRAS), which has the natural advantages of efficient lipid synthesis and protein secretion (Larroude et al. 2018). In recent years, sequencing technologies and gene editing technologies are widely known (Wagner et al. 2018). Y. lipolytica also has a clear genetic background, mature gene editing tools and abundant acetyl-CoA in its body. Therefore, Y. lipolytica is an ideal host cell for omega-3 fatty acid production.

Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major mechanisms of DNA damage repair (Wright et al. 2018). The former is often used for knockout and targeted integration of target genes, while the latter is often used for random integration of target genes. Specifically, homologous recombination mechanisms are used for knockout or targeted integration of target genes. DNA fragments containing upstream and downstream homologous arms, screening markers and target genes are constructed by PCR. Then the DNA were introduced into the competent cell of yeast to integrate into the corresponding genome. Then the correct transformants were obtained and the screening marker URA3 was recovered in 5-Fluoroorotic acid (5-FOA) solid medium. Unlike targeted integration of genes, random integration of genes does not require homologous arms. The exogenous DHA fragment is randomly inserted into the microbial genome using a non-homologous end-joining mechanism in host.

There are two natural polyunsaturated fatty acids (PUFAs) biosynthesis pathways in microorganisms, namely the anaerobic polyketide synthase (PKS) pathway, and the aerobic desaturase/ elongase pathway. Since the detailed biosynthesis mechanism of EPA synthesized by the PKS pathway is still unclear, the latter one is the preferred pathway to be modified and applied in Y. lipolytica. However, the wild-type Y. lipolytica are naturally unable to synthesize omega-3 PUFAs, but only linoleic acid (LA, C18:2). Therefore, an omega-3 PUFAs biosynthesis pathway be artificially created in Y. lipolytica for EPA production. Y. Lipolytica naturally has two extension/ desaturase pathways, which are ∆-6 and ∆-9 pathways. Among them, the ∆-6 elongation/desaturase pathway has a variety of by-products, so the latter is selected.

The gene of PEX10 is involved in the introduction of peroxisome matrix proteins and peroxisome proliferation. In addition, PEX10 is also involved in the β-oxidation pathway of fatty acids. Thus, the PEX10 was knocked-out results in high EPA titers and may increase the production of other desirable lipid-related products (Wei et al. 2021; Xue et al. 2013).

Recently, coupling precursor overproduction and driving forces with a metabolic sink to enable a push and pull dynamic regulation has become a very powerful strategy in metabolic engineering (Wang et al. 2022). Specifically, based on the higher content of intermediate fatty acids in the engineered strain, the next catalytic step gene is overexpressed and the metabolic flux is pushed downward. Or overexpression of upstream genes with lower content of intermediate fatty acids, metabolic flux is pulled down from top to bottom. In summary the push-pull strategy aims to increase the metabolic flux of the target product and improve the titer of the compound.

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Wagner JM, Williams EV, Alper HS (2018) Developing a piggyBac Transposon System and Compatible Selection Markers for Insertional Mutagenesis and Genome Engineering in Yarrowia lipolytica. Biotechnol J 13(5):e1800022 doi:10.1002/biot.201800022

Wang K, Shi TQ, Wang J, Wei P, Ledesma-Amaro R, Ji XJ (2022) Engineering the Lipid and Fatty Acid Metabolism in Yarrowia lipolytica for Sustainable Production of High Oleic Oils. ACS Synth Biol 11(4):1542-1554 doi:10.1021/acssynbio.1c00613

Wei LJ, Cao X, Liu JJ, Kwak S, Jin YS, Wang W, Hua Q (2021) Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2. Appl Environ Microbiol 87(17):e0048121 doi:10.1128/AEM.00481-21

Wright WD, Shah SS, Heyer WD (2018) Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem 293(27):10524-10535 doi:10.1074/jbc.TM118.000372

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