Seed-Specific Heterologous Expression of a Nasturtium FAE Gene in Arabidopsis Results in a Dramatic Increase in the Proportion of Erucic Acid

Abstract

The fatty acid elongase [often designated FAE or b-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium b-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.Very long chain fatty acids (VLCFAs) with 20 carbons or more are widely distributed in nature. In plants, they are mainly found in epicuticular waxes and in the seed oils of a number of plant species, including members of the Brassicaceae, Limnantheceae, Simmondsia, and Tropaeolaceae (Harwood, 1996;Post-Beittenmiller, 1996;Ghanevati and Jaworski, 2001). A strategic goal in oilseed modification is to genetically manipulate high erucic acid germ plasm of the Brassicaceae to increase the content of erucic acid (22:1 D13) and other strategic VLCFAs in the seed oil for industrial niche market needs (Puyaubert et al., 2001;Taylor et al., 2001). Erucic acid and its derivatives are feedstocks in manufacturing slip-promoting agents, surfactants, plasticizers, nylon 1313, and surface coatings; more than 1,000 patents have been issued (Sonntag, 1991(Sonntag, , 1995Leonard, 1994). The current market for high erucate oils exceeds $120 million U.S./annum. Worldwide erucic acid demand is predicted to increase from about 40 million pounds (M pds) in 1990 to about 80 M pds by the year 2010. Similarly, demand for the derivative, behenic acid, is predicted to triple to about 102 M pds by 2010 (Sonntag, 1995). In recent years, production has increased to meet market needs, and high erucic acreage in western Canada is currently at a record high (D. Males, Saskatchewan...

Saturated Very-Long-Chain Fatty Acids Promote Cotton Fiber and Arabidopsis Cell Elongation by Activating Ethylene Biosynthesis

“…Gh KCS13/CER6 shares 85 and 55% amino acid sequence identity with At CER6 (At CUT1) and Tropaeolum majus NasFAE, respectively. The latter enzyme was found to produce C22 from C20 (Mietkiewska et al, 2004), similar to the finding in this work. The enzyme activities of At CER6 (At CUT1) were never biochemically characterized.…”

Novel Insights into Seed Fatty Acid Synthesis and Modification Pathways from Genetic Diversity and Quantitative Trait Loci Analysis of the Brassica C Genome

“…A low 22:1n9 phenotype can be attributed to a single amino acid substitution, and has been demonstrated by substituting FAE1-Phe-282 with Ser (Katavic et al, 2002). Supporting evidence for specificity of FAEs has also been observed in Nasturtium (Tropaeolum majus), where an FAE gene was found to have a strong preference for elongation of 20:1-CoA (Mietkiewska et al, 2004). Selecting for reduced enzyme activity that modulates sequestration into pathway D could be used to further modulate the desired composition of rapeseed oil.…”

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar

“…In yeast, PotriKCS1 elongates monounsaturated and saturated FAs, whereas PotriKCS2 preferentially elongates saturated FAs. This ability to elongate saturated and monounsaturated FAs is shared by other KCSs (Kunst et al, 1992;Mietkiewska et al, 2004;Tresch et al, 2012).…”