Carlene Sarvas1, Debbie Puttick1, Li Forseille2, Dustin Cram2, Mark A Smith3. 1. Linnaeus Plant Sciences, 2024-110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada. 2. National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada. 3. Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada. Mark.Smith2@Canada.ca.
Abstract
MAIN CONCLUSION: A β-ketoacyl-ACP-synthase II (KAS2) like enzyme and a lysophosphatidic acid acyltransferase (LPAT2) from Consolida ajacis catalyze gondoic acid biosynthesis and incorporation into the sn-2 position of seed TAG in engineered Camelina sativa. Gondoic acid (cis-11 eicosenoic acid, 20:1∆11) is the predominant very-long-chain fatty acid (VLCFA) in camelina (Camelina sativa) seed oil accounting for 12-15% of total triacylglycerol fatty acids. To explore the feasibility of engineering increased levels of this fatty acid in camelina seed, oils from a range of plant species were analyzed to identify those producing 20-Carbon (C20) fatty acids as the only VLCFAs in their seed oil. Seeds of Consolida and Delphinium species (Ranunculaceae) were found to contain moderate levels (0.2% to 25.5%) of C20 fatty acids without accompanying longer chain fatty acids. The C20 fatty acids were abundant in both sn-2 and sn-1/3 positions of seed TAG in Consolida, but were largely absent from the sn-2 position in Delphinium seed TAG. Through generation of a developing seed transcriptome, sequences were identified and cDNAs amplified from Consolida ajacis encoding a β-ketoacyl-ACP-synthase II like protein (CaKAS2B) that lacked a predicted chloroplast transit peptide, and two homologues of Arabidopsis thaliana lysophosphatidic acid acyltransferase 2 (CaLPAT2a and CaLPAT2b). Expression of CaKAS2B in conventional (WT) camelina and a line previously engineered for high seed oleic acid content (HO) resulted in increased seed VLCFA content. Total VLCFA levels were raised from 24 to 35% and from 7 to 23% in T3 seed from representative transformants in the WT and HO backgrounds, respectively. Gondoic acid was the predominant VLCFA in transformed HO lines with low endogenous cytoplasmic fatty acid elongation activity, suggesting limited capacity of CaKAS2B to elongate beyond C20. Expression in camelina of CaLPAT2b resulted in significantly increased C20-VLCFA esterification at the sn-2 position of seed TAG with VLCFA levels of 33.8% in this position in one transformed line compared to 0.3% at sn-2 in the corresponding control line. Only small changes in total seed VLCFA content were observed in transformed lines implying that increased VLCFA esterification capacity in camelina results in positional redistribution of VLCFAs but does not significantly enhance flux through the fatty acid elongation pathway. The full potential of CaKAS2B and CaLPAT2a for the engineering of high gondoic acid levels in camelina remains to be determined. Seed fatty acid composition of Consolida and Delphinium also provides information that may be of value in the systematics of the Ranunculaceae.
MAIN CONCLUSION: A β-ketoacyl-ACP-synthase II (KAS2) like enzyme and a lysophosphatidic acid acyltransferase (LPAT2) from Consolida ajacis catalyze gondoic acid biosynthesis and incorporation into the sn-2 position of seed TAG in engineered Camelina sativa. Gondoic acid (cis-11 eicosenoic acid, 20:1∆11) is the predominant very-long-chain fatty acid (VLCFA) in camelina (Camelina sativa) seed oil accounting for 12-15% of total triacylglycerol fatty acids. To explore the feasibility of engineering increased levels of this fatty acid in camelina seed, oils from a range of plant species were analyzed to identify those producing 20-Carbon (C20) fatty acids as the only VLCFAs in their seed oil. Seeds of Consolida and Delphinium species (Ranunculaceae) were found to contain moderate levels (0.2% to 25.5%) of C20 fatty acids without accompanying longer chain fatty acids. The C20 fatty acids were abundant in both sn-2 and sn-1/3 positions of seed TAG in Consolida, but were largely absent from the sn-2 position in Delphinium seed TAG. Through generation of a developing seed transcriptome, sequences were identified and cDNAs amplified from Consolida ajacis encoding a β-ketoacyl-ACP-synthase II like protein (CaKAS2B) that lacked a predicted chloroplast transit peptide, and two homologues of Arabidopsis thaliana lysophosphatidic acid acyltransferase 2 (CaLPAT2a and CaLPAT2b). Expression of CaKAS2B in conventional (WT) camelina and a line previously engineered for high seed oleic acid content (HO) resulted in increased seed VLCFA content. Total VLCFA levels were raised from 24 to 35% and from 7 to 23% in T3 seed from representative transformants in the WT and HO backgrounds, respectively. Gondoic acid was the predominant VLCFA in transformed HO lines with low endogenous cytoplasmic fatty acid elongation activity, suggesting limited capacity of CaKAS2B to elongate beyond C20. Expression in camelina of CaLPAT2b resulted in significantly increased C20-VLCFA esterification at the sn-2 position of seed TAG with VLCFA levels of 33.8% in this position in one transformed line compared to 0.3% at sn-2 in the corresponding control line. Only small changes in total seed VLCFA content were observed in transformed lines implying that increased VLCFA esterification capacity in camelina results in positional redistribution of VLCFAs but does not significantly enhance flux through the fatty acid elongation pathway. The full potential of CaKAS2B and CaLPAT2a for the engineering of high gondoic acid levels in camelina remains to be determined. Seed fatty acid composition of Consolida and Delphinium also provides information that may be of value in the systematics of the Ranunculaceae.
Authors: Anders S Carlsson; Samuel T LaBrie; Anthony J Kinney; Penny von Wettstein-Knowles; John Browse Journal: Plant J Date: 2002-03 Impact factor: 6.417