Isolation of a Δ5 Desaturase Gene from Euglena gracilis and Functional Dissection of Its HPGG and HDASH Motifs
Delta (Δ) 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 Δ5 desa...
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Published in | Lipids Vol. 47; no. 9; pp. 913 - 926 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Berlin/Heidelberg
Springer-Verlag
01.09.2012
Springer‐Verlag |
Subjects | |
Online Access | Get full text |
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Summary: | Delta (Δ) 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 Δ5 desaturase gene from
Euglena gracilis
(
EgΔ5D
) 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
EgΔ5D
was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that
EgΔ5D
has about 39 % identity with a Δ5 desaturase of
Phaeodactylum tricornutum
. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing
Yarrowia lipolytica
strain,
EgΔ5D
had strong Δ5 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
EgΔ5D.
A double mutant
EgΔ5D
-
34G158G
with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type
EgΔ5D
. Codon optimization of the N-terminal region of
EgΔ5D
-
34G158G
and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %. |
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Bibliography: | The online version of this article (doi:10.1007/s11745‐012‐3690‐1) contains supplementary material, which is available to authorized users. Electronic supplementary material ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0024-4201 1558-9307 |
DOI: | 10.1007/s11745-012-3690-1 |