Characterization of the Brassica napusExtraplastidial Linoleate Desaturase by Expression in Saccharomyces cerevisiae  

Abstract

The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system. A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids. The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures. The results indicate that the enzyme has: (a) -3, as opposed to ⌬-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with -6 double bonds, but the ability to desaturate substrates with -6 hydroxyl groups or -9 or -5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate.

Two FAD3 Desaturase Genes Control the Level of Linolenic Acid in Flax Seed

“…This contrasts with the result reported by Reed et al (2000) where desaturation of both of these fatty acids occurred in cultures of yeast transformed with the B. napus FAD3 gene. However, desaturation levels observed with the Brassica gene were extremely low, and it is probable that conditions in the experiments reported here did not allow us to detect such low levels of activity.…”

“…In control samples, endogenous 18:1n-9 was not desaturated. However, in samples carrying the Normandy LuFAD3 cDNAs without exogenous substrates, a small peak corresponding to the expected position of 18:2 (9,15) was detected (data not shown), which likely resulted from the desaturation of endogenous 18:1n-9; this desaturation activity was also detected with a Brassica napus FAD3 gene (Reed et al, 2000). Although 18:2(9,15) has a slightly longer retention time than does 18:2n-6, it is not detectable in samples from cultures fed 18:2n-6 because its peak overlaps with the very broad linoleic acid peak.…”

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

“…Because the polyunsaturated products of the -3 and "⌬12" desaturases are not metabolized further, the final levels of product in the total lipid extract may be used as a semiquantitative indicator of enzyme function (21,28). For functional characterization, the wild type and chimeric enzymes were expressed in the S. cerevisiae strain INVSc1 supplied with various fatty acid substrates, and the recombinant yeast cells were analyzed for fatty acid content.…”

Activity of Brassica napus and Perilla frutescens microsomal ω-3 desaturases expressed in yeast (Saccharomyces cerevisiae)

“…In addition, it shows ω-3 regioselectivity Meesapyodsuk et al, 2000). Expression of plant desaturases in yeast offers a rapid method of verifying the enzymes' activity as well as the characteristics of their relationships with their substrates and the products, in addition to the study of their behaviors under different physiological conditions such as the effect of temperature on the accumulation of different fatty acids in storage lipids (Covello and Reed, 1996;Reed et al, 2000;Cahoon et al, 2001;Dyer et al, 2001;Sayanova et al, 2001;Hong et al, 2002;Sandager et al, 2002;Sharma et al, 2012). The yeast expression model may provide useful information for understanding the transcriptional and posttranscriptional mechanisms involved in the regulation of plant fatty acid desaturases (Dyer et al, 2001), which in turn could provide tools to increase our ability to produce engineered plants containing nutritionally and industrially useful fatty acid compositions (Budziszewski et al, 1996;Kinney, 1997;Moon et al, 2000;Cahoon and Shanklin 2000;Dyer et al, 2001Dyer et al, , 2002.…”