Abstract
Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.
Highlights
Long-chain polyunsaturated fatty acids (LC-PUFAs), the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects
Due to its non-pathogenic nature, several processes based on Y. lipolytica were categorized as generally recognized as safe (GRAS)[5]
The crucial difference between oleaginous and non-oleaginous yeasts becomes evident during cultivation under nitrogen-limiting conditions[7,8]: the carbon flux in non-oleaginous yeasts is directed into synthesis of various polysaccharides, whereas in oleaginous yeasts, it is preferentially channeled towards lipid biosynthesis, leading to an accumulation of triacylglycerols within discrete intracellular lipid bodies
Summary
Long-chain polyunsaturated fatty acids (LC-PUFAs), the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains. As already demonstrated in a previous study, expression of a bifunctional Δ12/ω3 desaturase from Fusarium moniliforme in Y. lipolytica yielded α-linolenic acid (18:3, n-3), a precursor of EPA and DHA, at a concentration of 28.1% of total fatty acids (TFAs)[10]. A Y. lipolytica strain that produces EPA at 56.6% of TFAs was generated by DuPont (USA)[12] These production levels were reached by overexpression of 30 copies of nine different, codon-optimized genes (20 desaturase genes, eight elongase genes, and two cholinephosphotransferase genes) combined with disruption of four genes, including a gene encoding a peroxisomal biogenesis factor and two genes involved in the lipid metabolism, in the yeast genome
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