Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants Guohai Wu 1 , Martin Truksa 1 , Nagamani Datla 1 , Patricia Vrinten 1 , Joerg Bauer 2 , Thorsten Zank 3,4 , Petra Cirpus 2 , Ernst Heinz 3 & Xiao Qiu 1 Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits 1–5 . We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important x-3 fatty acid in oilseed crops. Plants have the capacity to serve as green factories for the production of novel industrial materials, nutritionally enhanced foods or phar- maceuticals, via metabolic engineering 6–8 . One goal of plant metabolic engineering is the production of high levels of VLCPUFAs in oilseed plants 9,10 , which would provide a novel and cost-effective source of these fatty acids. Several pathways for the biosynthesis of VLCPUFAs exist in nature 11 . To produce VLCPUFAs in seeds, we followed the alternating desaturation/elongation pathways of n-6 and n-3 fatty acids. The two routes commence with linoleic acid (18:2n-6, LA) and a-linolenic acid (18:3n-3, ALA), respectively, followed by sequential D6 desaturation, D6 elongation and D5 desaturation, leading to the synthesis of arachidonic acid (20:4n-6, AA) in the n-6 and eicosapentaenoic (20:5n-3, EPA) in the n-3 pathway. The two pathways can be interconnected by a o3 desaturase that converts AA into EPA. Further D5 elongation and D4 desaturation reactions lead to the synthesis of docosapentaenoic (22:5n-3, DPA) and finally doc- osahexaenoic acid (22:6n-3, DHA). The B. juncea breeding line 1424 was chosen as a host plant for biosynthesis of VLCPUFAs because of its high LA content and lack of erucic acid. The constructs for VLCPUFA production in seeds carried three to nine structural genes, with each gene under the control of the seed-specific napin promoter (Fig. 1). The first construct (BJ3) introduced into B. juncea contained a D6 desaturase from Pythium irregulare 12 ,a D5 desaturase from Thraus- tochytrium sp. 13 and a D6 fatty acid elongase from Physcomitrella patens 14 . This represents the minimal set of transgenes required for the synthesis of AA and EPA from endogenous LA and ALA. RT-PCR indicated that all three genes were highly expressed in the developing seeds (data not shown). Several new fatty acids were detected in BJ3 seeds (Fig. 2). The most abundant was g-linolenic acid (GLA), the D6 desaturation product of LA, with an average value of 27.7% of total seed fatty acids. AA, the D5 desaturation product of dihomo-g- linolenic acid (20:3n-6, DGLA), ranged from 5.0% to 8.5% (average 7.3%), whereas stearidonic acid (SDA), the D6 desaturation product of ALA, averaged 3.1%; several other minor new fatty acids, such as 18:2n-9 (1.7%), were also present (Table 1). Consequently, LA content dropped dramatically from 45.2% in the untransformed control to 13.7% in transgenic seeds. Thus, the D6 and D5 desaturases func- tioned well, with conversion rates of 68.3% and 94.2%, respectively. The D6 elongase performed less efficiently, with a conversion rate of only 23.6%. The n-6 pathway appeared to be much more effective in VLCPUFA biosynthesis, perhaps not surprisingly, given that B. juncea oil is characterized by high LA (45.2%) and low ALA (9.7%). To increase LA and concurrently reduce the side-product 18:2n-9, we added a D12 desaturase gene from Calendula officinalis 15 to the triple construct, producing the construct BJ4. The addition of this desaturase resulted in only a slight decrease (0.5%) of 18:2n-9. Enhanced conversion of oleic acid (OA) to LA was evident from the decrease in OA content. Although the GLA content remained similar to that in BJ3 plants, the average level of AA increased from 7.3% to 12.0%, with the highest level reaching 17.7%. EPA also increased from 0.8% to 1.3% (Table 1). In view of the results from the BJ3 and BJ4 plants, where poor elongation from 18- to 20-carbon fatty acids limited the meta- bolic flux, we attempted to enhance elongation by adding a second D6 elongase from Thraustochytrium sp. When expressed in yeast, this elongase showed activity with both 18-carbon and 20-carbon fatty acids, but elongated GLA and SDA much more efficiently than AA and EPA (data not shown). In transgenic plants carrying this construct, named BJ5, a slight, but still significant increase in GLA elongation occurred. This in turn resulted in an increase in AA from an average of Published online 12 June 2005; doi:10.1038/nbt1107 1 Bioriginal Food & Science Corporation, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9. 2 BASF Plant Science GmbH, 67117 Limburgerhof, Germany. 3 Biozentrum Klein Flottbek, Universita ¨t Hamburg, 22609 Hamburg, Germany. 4 Present address: BASF Plant Science GmbH, 67117 Limburgerhof, Germany. Correspondence should be addressed to X.Q. (xqiu@bioriginal.com). NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 8 AUGUST 2005 1013 LETTERS © 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology