Identification and characterization of new Δ-17 fatty acid desaturases

Abstract

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

“…This was then divided into six 25-ml cultures. After incubation for 48 h at 30°C at 250 rpm, a sample from the first time point (day 0) was taken for dry cell weight (DCW) and fatty acid analysis, as described previously (23)(24)(25). Briefly, a 1-ml aliquot was collected by centrifugation, and lipids were extracted.…”

Characterization and molecular docking of new Δ17 fatty acid desaturase genes from Rhizophagus irregularis and Octopus bimaculoides

“…The typical types of n3Des have been reported from a wide range of species including microalgae, cyanobacteria, yeast, fungi and plants. 6,22,34 The phylogenetic tree in Fig. 3 shows that the top group of n3Des was derived from M. alpina, yeast and plants.…”

“…[3][4][5] However, the limited natural sources of n-3 PUFAs come mainly from microalgae oil and sh oil. 6 As an alternative source, microorganisms have been used to produce EPA and DHA. 7 The n-6/n-3 fatty acid metabolic ux in microbial cells is tightly regulated by its intrinsic synthesis pathways (D6Des/ D6Elo/n3Des or D9Elo/D8Des/n3Des), and the key regulator n3Des is located at the branch point of metabolic ux.…”

“…10,11 Some reports have involved studies of various types of n3Des from a wide range of species including microalgae, cyanobacteria, yeast, fungi and plants. 6,7,12 n3Des from various species were shown to exhibit a broad n-6 fatty acid substrate specicity with different catalytic efficiencies and preferences in the presence of various fatty acid substrates. 13 The rst yeast n3Des gene cloned from Saccharomyces kluyveri showed a dramatic preference for LA over AA substrate, 14,15 and the same preference was found in the bifunctional D12/n3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea.…”

Metabolic engineering of Yarrowia lipolytica for industrial applications

“…First, an efficient EPA biosynthetic pathway was built using strong promoters, codon-optimized heterologous genes, and multiple gene copies for each step. Second, the carbon flux was pushed toward the EPA biosynthesis pathway by over-expression of C16E [50] and D12D [52], and was pulled for EPA biosynthesis by using multiple copies of D17-desaturase genes [53]. Third, b-oxidation was eliminated by targeted deletion of peroxin genes [37 ,54].…”