The Enzymology of Castor Oil Biosynthesis

The castor plant Ricinus communis L., Euphorbiaceae produces seeds containing a unique oil. Castor oil is the only commodity vegetable oil that contains significant amounts of hydroxy fatty acid. This fatty acid is ricinoleic acid (12-hydroxy oleic acid) (Fig. 1) and it comprises up to 90% of the total fatty acid (FA) content of castor oil. Due to the physical and chemical properties imparted by the mid-chain hydroxy group, the oil has many important industrial uses. However, due to the presence of the toxic protein ricin and hyperallergenic 2S albumins, the production of castor oil is problematic. One logical approach to solving the biohazard problem presented by castor meal is to produce ricinoleate in plants lacking these noxious components. Several researchers have attempted to produce a ricinoleate oil by transgenic expression of the oleoyl-12-hydroxylase, the enzyme that is responsible for ricinoleate biosynthesis. This approach of introducing a single gene to engineer a novel oil composition was pioneered by the Calgene company the in order to develop laurate canola (Voelker et al. 1992). Expression of the cDNA that encodes the oleoyl-12-hydroxylase (or FAH, for fatty acyl hydroxylase), resulted in the accumulation of a low level of hydroxy fatty acids in tobacco and Arabidopsis (Broun and Somerville 1997). These results suggested that the FAH gene by itself is not sufficient to produce high levels of ricinoleate in plants other than castor (McKeon and Lin 2002). An in vitro system using microsomes isolated from developing castor seed endosperm provided an effective means for following fatty acid hydroxylation and castor oil biosynthesis (McKeon et al. 1997; Lin et al. 1998a). Using metabolic profiling tools for analyzing lipid biosynthetic products (Lin et al. 1998a, 2002), the analysis of radiolabeled products from fatty acids incubated in the microsomal system enabled identification of several enzyme activities that give castor its unique ability to produce a high ricinoleate oil (McKeon and Lin 2002). Based on these studies, the preferential incorporation of ricinoleate into triacylglycerol (TG) led us to identify the final step in oil biosynthesis (Fig. 2) as a key step in maintaining high ricinoleate content while minimizing oleate incorporation into the TG fraction. Table 1 shows that the microsomes incorporate ricinoleate preferentially by a factor of 6-fold. The diacylglycerol acyltransferase (DGAT) is a transmembrane enzyme that catalyzes the acylation of diacylglycerol (DG) to TG, using acylCoA as the source for the final acyl group. This step has long been considered to be a rate limiting step in oil biosynthesis, and considerable evidence has accumulated to indicate that altered DGAT activity levels dramatically affect the yield of oil (He et al. 2004). AcylCoA synthetases (ACS) produce the acyl-donor