Identification and characterization of new Δ-17 fatty acid desaturases.

Zhixiong Xue,Hongxiang Zhang,Daniel J Macool,Bogdan Szostek,Dieter Hollerbach,Hongxian He,Narendra S Yadav,Quinn Zhu

doi:10.1007/s00253-012-4068-2

Zhixiong Xue, Hongxiang Zhang + Show 6 more

Open Access

https://doi.org/10.1007/s00253-012-4068-2

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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.

Highlights

Long-chain polyunsaturated fatty acids (LCPUFAs), especially ω-3 LCPUFAs eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are essential nutrients critical to human nutrition and health
This suggests that the P. aphanidermatum strain has a complete synthetic pathway for EPA, including both a Δ-5 desaturase and a Δ-17 desaturase
To broaden the search for Δ-17 desaturases, we looked for this enzyme in other oomycetes species by using the amino acid sequence of PaD17 as a query to BLAST the P. sojae and P. ramorum databases of Department of Energy’s Joint Genome Program

Summary

Introduction

Long-chain polyunsaturated fatty acids (LCPUFAs), especially ω-3 LCPUFAs eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are essential nutrients critical to human nutrition and health These fatty acids cannot be synthesized de novo in mammals and so must be obtained either directly through diet or indirectly through further desaturation and elongation of other polyunsaturated fatty acids (PUFAs) widely available in the diet, such as linoleic acid (LA) or α-linolenic acid (ALA) (Holman 1986; Lands 1992; Bézard et al 1994; Moghadasian 2008). LCPUFAs are part of the cellular membrane system and play essential roles in determining the structure and function of the membrane (Rabinovich 1991; Stillwell and Wassall 2003; Ma et al 2004) They are necessary for proper development in mammals, in the developing infant brain, and for tissue formation and repair. Numerous clinical studies have documented wide-ranging health benefits conferred by administration of ω-3 LCPUFAs against a variety of symptoms and diseases such as asthma, cancer, depression, diabetes, immune disorder, and skin conditions (Mickleborough et al 2006; Colomer et al 2007; Damsgaard et al 2007; Amminger et al 2010; Wall et al 2010; Djoussé et al 2011)

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