Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana

Abstract

Expression of a cDNA encoding the castor bean ( Ricinus communis L.) oleate Delta12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec- cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec- cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos- cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos- cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Delta-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Delta12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Delta12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

“…Our results with RcROD1 in different transgenic lines, along with previous reports (Smith et al, 2003;Lu et al, 2006;Burgal et al, 2008;van Erp et al, 2011), point out the complexity of the interactions among different mechanisms that channel HFAs into TAG. PDCT enhances the acyl fluxes through PC and thus reduces the pathway toward elongation outside of PC.…”

“…The elongation pathway is blocked in the CL37 and CL7-RcDGAT2 lines that were generated in the fae1 background. The CL37 seeds already contain 17% HFAs, which is substantially higher than the levels in the Col-FAH12 lines in this study and the lines reported previously (Smith et al, 2003). However, RcROD1 still significantly increases HFA levels in the CL37 and CL7-RcDGAT2 lines (Table II).…”

“…The FA composition of transgenic red seeds was determined by analyzing fatty acid methyl esters (FAMEs) using gas chromatography (GC). Four novel FAs were detected compared with wild-type seeds, which corresponded to the HFAs previously identified as 18:1OH, densipolic acid (18:2OH), lesquerolic acid (20:1OH), and a small amount of auricolic acid (20:2OH; Broun and Somerville, 1997;Smith et al, 2003). The total HFA contents in each of the transgenic lines ranged from 4% to 5% in the rod1 mutant, compared with approximately 10% in the wild-type background (Fig.…”

“…The 18:1OH is produced by the hydroxylation of 18:1 that is esterified to the sn-2 position of PC (Bafor et al, 1991) by the oleate D12-hydroxylase (FAH12; van de Loo et al, 1995). Seed-specific expression of FAH12 in Arabidopsis or the oilseed crop camelina (Camelina sativa) only resulted in up to approximately 17% total HFAs including 18:1OH and its derivatives (Broun and Somerville, 1997;Smith et al, 2003;Lu et al, 2006;Lu and Kang, 2008). Coexpressing a PDAT (RcPDAT1A) from castor with FAH12 significantly increased the HFA levels up to approximately 27% (van Erp et al, 2011).…”

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

“…Among many attempts, an alkaline pretreatment is known to efficiently remove lignin from plant cell wall structures [11]. Cellulose and hemicellulose are depolymerized to monosaccharides by synergetic actions of enzyme mixtures [12]. The most abundant monomer sugars derived from lignocellulosic biomass are D-glucose, since cellulose represents 70% of total plant cell walls, repeating the β-(1→4) glycosidic bond [13].…”