Metabolic Engineering of Vegetable Oils withVery Long-Chain Fatty Acids (Vlcfas)

Although the fatty acyl synthase cannot produce fatty acids longer than 18 carbons, extraplastidial complexes known as acyl-CoA elongases can extend fatty acids to 20, 22, or even double those numbers of carbons. While all plants require VLCFAs for production of the cuticle and extracuticular wax, some incorporate them into seed oils. For example, erucic acid (22:1Δ13) made by the elongation of oleic acid (18:1Δ9) by two C2 units is an industrially useful fatty acid found in rapeseed oil as well as the model oilseed A. thaliana.

The acyl-CoA elongase performs reactions comparable to those of the fatty acyl-ACP synthase, employing KCS, 3-ketoacyl-CoA reductase, 3-hydroxyacyl dehydratase and enoyl reductase subunits. Of the elongase components, the KCS appears to be rate limiting (Ghanevati and Jaworski, 2002; Millar and Kunst, 1997). For example, the production of low erucic acid canola oil from high erucic acid varieties of rapeseed was made possible by a single amino acid change in a KCS (Katavic et al., 2002). Conversely, overexpression of the A. thaliana FAE gene, whose KCS product initiates the elongation cycle in A. thaliana, results in production of fatty acids up to 22 carbons long in organs that do not normally accumulate them (Millar et al., 1998). In addition, the introduction of a KCS from L. douglasii into soybean somatic embryos resulted in the accumulation of C20 and C22 fatty acids to amounts of >15% of the total fatty acids (Cahoon et al., 2000). These fatty acids normally comprise <1% of the fatty acids of this tissue. A variety of KCS polypeptides with differing substrate specificities have been identified (Cahoon et al., 2000; Lassner et al., 1996; Millar et al., 1999; Moon et al., 2001).

Evidence indicates that KCS may not be the only enzyme class capable of initiating VLCFA synthesis in plants. For example, elongation of C18 Δ6-polyunsaturated fatty acids in the moss P. patens is initiated by an enzyme with homology to a class of polypeptides termed ELOs (Zank et al., 2002). These enzymes were first identified as components of the fatty acid elongation system in S. cerevisiae (Toke and Martin, 1996). and have been characterized from a variety of animal and fungal species (Beaudoin et al., 2000; Parker-Barnes et al., 2000). Genes for ELO-like enzymes are also present in A. thaliana and other plants, but their functions have yet to be determined. The ELO polypeptides contain the motif HXXHH, which is similar to a sequence element found in fatty acid desaturases (Parker-Barnes et al., 2000). Whether these histidines coordinate active site iron atoms, as they do in desaturases, is not known. The ELOs from organisms such as C. elegans and the fungus M. alpina participate in the synthesis of nutritionally important long-chain polyunsaturated fatty acids (Beaudoin et al., 2000; Parker- Barnes et al., 2000). ELO and KCS enzymes appear functionally redundant with regard to their ability to catalyze the first step in fatty acid elongation, yet these enzyme classes are structurally unrelated.

Enrichment of TAG with VLCFAs might be limited by the ability of plants to incorporate these unusual molecules into glycerolipids. For example, rapeseed, although it does accumulate 22:1Δ13, is unable to incorporate this fatty acid into the sn-2 position. However, if the plants are transformed with a Limnanthes alba gene for an LPAAT utilizing 22:1Δ13-CoA, some tri-22:1Δ13 is produced (Lassner et al., 1995). Similarly, a yeast LPAAT, designated SLC1–1, that has specificity for C22 and C24 acyl-CoAs was shown to improve both TAG yield and proportions of VLCFAs when expressed in B. napus seeds (Zou et al., 1997).