Optimisation of trierucin content in oilseed rape (2005)

High erucic acid rapeseed (HEAR) cultivars have regained interest for industrial purposes because erucic acid (22:1) is a valuable feedstock with a wide range of applications especially if it is available in sufficient quantities and in sufficiently pure form. In HEAR oil, however, the 22:1 content is limited to 67% because 22:1 is excluded from the sn-2 position of the glycerol backbone. To overcome this theoretical limit in HEAR oil and, thus, to improve its oleochemical properties combined efforts in plant breeding and genetic engineering have been undertaken. Co-expression of an 22:1-CoA specific lysophosphatidate acyltransferase gene with a ß-ketoacyl-CoA synthase (KCS) gene in developing seeds of HEAR plants resulted in the channeling of 22:1 into each position of the glycerol backbone but hardly improved the total 22:1 content. The enzymic investigations with microsomal fractions of developing seeds conducted in the present study clearly showed that the introduced KCS gene was functionally expressed in the developing seeds of the transgenic HEAR plants and affected a threefold increase in KCS as well as elongase activity catalyzing the malonyl-CoA dependent elongation of oleoyl(18:1)-CoA via eicosenoyl(20:1)-CoA to 22:1-CoA. These data provide further evidence that KCS rather than the further three enzymes of the elongase systems catalyzes the rate limiting step. Analysis of the acyl-CoA and fatty acid composition in the course of seed development, however, revealed that during the phase of maximal oil accumulation the improved elongase activity of the developing seeds from the transgenic HEAR plants was no longer reflected in their 22:1 levels. These data suggest that the elongase activity and, thus, the formation rate of 22:1-CoA is limited by other factors such as the substrate pools available to the elongase complex. In order to improve the cytosolic acetyl-CoA and malonyl-CoA pool, chimeric gene constructs encoding ATP:citrate lyase and acetyl-CoA carboxylase were developed and introduced into HEAR lines. An overall increase in the 22:1 content of seed oil from transgenic plants was achieved by the over-expression of the chimeric acetyl-CoA carboxylase gene. According to these data it is likely that a further increase in the 22:1 content can be achieved by the co-expression of the acetyl-CoA carboxylase gene with the ATP:citrate lyase, KCS and 22:1-CoA specific lysophosphatidate acyltransferase gene. In addition, the sink-source relationship is important for the increase of unusual fatty acids in seed oils. Oil yield is decisively determined by the activities of the diacylglycerol acyltransferase (DAGAT) which catalyzes the acylation reaction at the sn-3 position of diacylglycerol in the final step of TAG synthesis. Hence, to improve the source strength by over-expressing a DAGAT that effectively channels 22:1 into TAG, DAGAT cDNAs were cloned and functionally characterized from Brassica napus, Tropaeolum majus and Lesquerella lindheimeri, the seed oils of which almost exclusively carry very long chain acyl groups at the sn-3 position. Six cDNAs, namely three of B. napus, two of L. lindheimeri and one of T. majus, were analyzed. The DAGATs encoded by these cDNAs possess very similar molecular masses, and apart from their N-terminal hydrophilic regions, their sequences are very similar to each other. The analysis of the properties of the DAGATs over-expressed in yeast revealed differences between the isofunctional enzymes especially with regard to their acyl-CoA specificities and selectivities. The DAGAT from T. majus displayed the lowest and those from L. lindheimeri the highest preference for very long chain acyl groups. Moreover, in spite of the high sequence identity between the two DAGATs of L. lindheimeri, DAGAT1 showed a more pronounced specificity for 20:1-CoA and 22:1-CoA in comparison to 18:1-CoA than DAGAT2. In order to elucidate the molecular basis of the different acyl-CoA specificities of the two DAGATs from L. lindheimeri, initial domain swapping and site directed mutagenesis experiments were conducted. The analysis of the acyl-CoA specificities of the chimeric and mutated DAGATs compared to DAGAT1 and DAGAT2 provided evidence that the acyl-CoA binding domain within the N-terminal region and isoleucine 315 in the fatty acid binding domain, which is substituted by a threonine in DAGAT1, can essentially determine acyl-CoA specificity. Moreover, these experiments resulted in the development of a DAGAT with an improved 22:1-CoA specificity. Consequently, the respective sequence appears to be better suited for improving the 22:1 content of HEAR oil than the DAGAT2 sequence used for rapeseed transformation in the present study in combination with a B. napus KCS and a 22:1-CoA specific lysophosphatidate acyltransferase gene.