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

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 gamma-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 Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-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.

Figure 1

Gas chromatogram of fatty acid methyl esters (FAME) from the lipid fraction in yeast containing pYES2 or in yeast clone P-708-2. All yeast strains were grown in selective medium supplemented with exogenous GLA at a final concentration of 25 μM. The arrows indicate the substrate as well as the newly synthesized DGLA in the culture of yeast clone P-708-2. The front end of the chromatogram is not shown.

Figure 2

Comparison of the deduced amino acid sequence of the M. alpina GLELOp with yeast ELO2p. Identical residues are shaded. The conserved histidine box is underlined.

Figure 3

Substrate specificity of the M. alpina GLELOp. The SC334(pRPB2) culture was grown in the presence of indicated substrates at a final concentration of 25 μM. Percent conversion was calculated as [product/(substrate + product)] × 100.