Lysophosphatidic Acid Acyltransferase from Meadowfoam Mediates Insertion of Erucic Acid at the sn-2 Position of Triacylglycerol in Transgenic Rapeseed Oil

Abstract

Lysophosphatidic acid acyltransferase acylates the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid, a precursor to triacylglycerol. A cDNA encoding lysophosphatidic acid acyltransferase was isolated from developing seeds of meadowfoam (Limnanthes alba alba). The cDNA encodes a 281-amino acid protein with a molecular m a s of 32 kD. The cDNA was expressed in developing seeds of transgenic high-erucic-acid rapeseed (Brassica napus) using a napin expression cassette. Erucic acid was present at the sn-2 position of triacylglycerols from transgenic plants but was absent from that position of seed oil extracted from control plants. Trierucin was present in the transgenic oil. Alteration of the sn-2 erucic acid composition did not affect the total erucic acid content. These experiments demonstrate the feasibility of using acyltransferases to alter the stereochemical composition of transgenic seed oils and also representa necessary step toward increasing the erucic acid content of rapeseed oil.

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

“…To overcome this limitation, the gene for an erucoyl-CoA preferring sn-2 acyltransferase was isolated from Limnanthes species. Expression of this gene in transgenic rapeseed altered seed oil sn-2 proportions of erucic acid, but did not lead to an increase in erucic acid content of the seed oil (Lassner et al 1995;Brough et al 1996;Weier et al 1997).…”

Erucic acid rapeseed: 1. Prospects of improvements

“…In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Knutzon et al, 1999;Lassner et al, 1995), a significant increase in the total amount of unusual fatty acid was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis thaliana with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al, 1997).…”

“…The same strategy has been used by Calgene (Lassner et al, 1995). The cDNA encoding LPAAT had been isolated from seeds of Limnanthes alba and was expressed in the seeds of transgenic rapeseed.…”

“…Limnanthes seed oil consists of more than 90% of VLCFAs (Lassner et al, 1995;Miller et al, 1964). The sn-2-position of Limnanthes douglasii, has a higher content of C22:1 relative to the total oil composition (Lassner et al, 1995;Phillips et al, 1971). Thus, cDNA encoding 1-acylglycerol-3-phosphate acyltransferase in L. douglasii was introduced into rape.…”

“…The first gene of LPAAT, plsC from E. coli, was cloned from a complementary homologous DNA, of a mutant strain of E. coli, which was deficient in the activity of LPA-AT (Coleman, 1990(Coleman, , 1992Frentzen, 1998). The gene of an erucoyl-CoA preferring sn-2 acyltransferase from Limnanthes douglasii (Ld-LPAAT) has been successfully cloned and overexpressed in rapeseed (Brough et al, 1996;Brown et al, 1995;Friedt and Lühs, 1998;Hanke et al, 1995;Lassner et al, 1995;Frentzen, 1998). However, the overall proportions of 22:1 in the seed oil did not increase.…”