Isolation of a Delta 5 Desaturase Gene from Euglena gracilis and Functional Dissection of Its HPGG and HDASH Motifs

• Published in: Lipids Vol. 47; no. 9; pp. 913 - 926
• Main Authors: Pollak, Dana Walters, Bostick, Michael W, Yoon, Hyeryoung, Wang, Jamie, Hollerbach, Dieter H, He, Hongxian, Damude, Howard G, Zhang, Hongxiang, Yadav, Narendra S, Hong, Seung-Pyo, Sharpe, Pamela, Xue, Zhixiong, Zhu, Quinn
• Format: Journal Article
• Language: English
• Published: 01.09.2012
• Subjects: Amino acids; Arachidonic acid; Arginine; Codons; desaturase; Docosahexaenoic acid; Eicosapentaenoic acid; Elongation; Enzymes; Euglena gracilis; Lipids; Nucleotides; Oligonucleotides; Phaeodactylum tricornutum; Polymerase chain reaction; Polyunsaturated fatty acids; Primers; Serine; Yarrowia lipolytica
• ISSN: 0024-4201; 1558-9307
• DOI: 10.1007/s11745-012-3690-1

Delta ( Delta ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the "desaturation and elongation" pathways. A full length Delta 5 desaturase gene from Euglena gracilis (Eg Delta 5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5' and 3' rapid amplification of cDNA ends. The whole coding region of Eg Delta 5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that Eg Delta 5D has about 39 % identity with a Delta 5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, Eg Delta 5D had strong Delta 5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of Eg Delta 5D. A double mutant Eg Delta 5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Delta 5 desaturase activity similar to the wild type Eg Delta 5D. Codon optimization of the N-terminal region of Eg Delta 5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.