Functional expression of Brassica juncea oleate desaturase gene (Bjfad2) in Escherichia coli

Abstract

The synthesis of polyunsaturated fatty acids, the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase-2 (FAD2; EC 1.3.1.35), also called as microsomal Δ12 oleate desaturase. The gene (Bjfad2; GenBank accession No. EF639848) coding for this enzyme from Brassica juncea was previously isolated and characterized. However, functional identity of Bjfad2 was not established. Utilizing the known Bjfad2 cDNA sequence, the ORF of Bjfad2 gene was cloned into the pMAL C2X Escherichia coli expression vector and produced recombinant plasmid by insertion of isolated ORF downstream to the maltose-binding protein coding sequence. The pMALC2X-Bjfad2 vector was used to transform the TB1 strain of E. coli. Induced expression of pMAL-BJFAD2 fused product resulted in the synthesis of a polypeptide with an apparent molecular mass of 80 kDa, which was 8 kDa less than calculated mass as determined by SDS-PAGE, since the fused MalE-Bjfad2 gene contains eight additional codons located between the MalE and Bjfad2 gene. In vitro activity assay of oleate desaturase using the corresponding bacterial crude extracts confirmed that the polypeptide was the product of the Bjfad2 gene. The reaction products analysis of the fatty acid methyl esters by gas chromatography showed the presence of a new peak with a similar retention time to linoleic acid, which was absent in the control activity assay without electron donors. Thus, B. juncea gene has been functionally identified since it encodes the enzyme that catalyzed the desaturation of oleate to linoleate.