Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.
Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.
Katavic
Vesna
V
Saskatchewan Wheat Pool Agricultural Research and Development, Saskatoon, Canada. Vesna.Katavic@nrc.ca
Mietkiewska
Elzbieta
E
Barton
Dennis L
DL
Giblin
E Michael
EM
Reed
Darwin W
DW
Taylor
David C
DC
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry; D000123:Acetyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D029688:Brassica napus / Q000201:enzymology* / Q000378:metabolism*; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D004883:Erucic Acids / Q000378:metabolism*; D000081031:Fatty Acid Elongases; D015151:Immunoblotting; D008861:Microsomes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012694:Serine / Q000737:chemistry; D013997:Time Factors
D004883:Erucic Acids; D010455:Peptides; C049811:erucic acid; D012694:Serine; D010649:Phenylalanine; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase