Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

Ross Products Division, Abbott Laboratories, Columbus, OH 43215

Edited by Christopher R. Somerville, Carnegie Institution of Washington, Stanford, CA, and approved May 26, 2000 (received for review November 22, 1999)

Abstract

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of γ-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Δ6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Δ5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

Footnotes

↵ * J.M.P.-B. and T.D. contributed equally to this work.
↵ † To whom reprint requests should be addressed. E-mail: pradip.mukerji{at}rossnutrition.com.
This paper was submitted directly (Track II) to the PNAS office.
The GLELO sequence reported in this paper has been submitted in the GenBank database (accession no. AF206662).
Abbreviations:

PUFA,

polyunsaturated fatty acid;

LA,

linoleic acid;

GLA,

γ-linolenic acid;

STA,

stearidonic acid;

DGLA,

dihomo-γ-linolenic acid;

AA,

arachidonic acid;

EPA,

eicosapentaenoic acid;

DOB,

dropout broth;

URA,

uracil;

LEU,

leucine;

MS,

mass spectrometry;

KCS,

β-ketoacyl-CoA synthase;

FAME,

fatty acid methyl ester