Linnaeus Plant Sciences, 2024-110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.
National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.
Planta. 2021 Jul 21;254(2):32. doi: 10.1007/s00425-021-03682-5.
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.
β-酮酰基-ACP 合酶 II（KAS2）样酶和来自 Consolida ajacis 的溶血磷脂酸酰基转移酶（LPAT2）催化了岗桐酸的生物合成，并将其掺入工程化的荠蓝种子 TAG 的 sn-2 位。岗桐酸（顺式-11 二十碳烯酸，20:1∆11）是荠蓝种子油中主要的超长链脂肪酸（VLCFA），占总三酰甘油脂肪酸的 12-15%。为了探索在荠蓝种子中增加这种脂肪酸水平的可行性，分析了一系列植物物种的油，以鉴定那些产生 20 碳（C20）脂肪酸作为其种子油中唯一的 VLCFAs 的物种。发现 Consolida 和 Delphinium 属（毛茛科）的种子含有中等水平（0.2%至 25.5%）的 C20 脂肪酸，而没有伴随更长链脂肪酸。C20 脂肪酸在 Consolida 的种子 TAG 的 sn-2 和 sn-1/3 位置都很丰富，但在 Delphinium 种子 TAG 的 sn-2 位置却很少。通过生成发育中的种子转录组，鉴定并扩增了来自 Consolida ajacis 的编码 β-酮酰基-ACP 合酶 II 样蛋白（CaKAS2B）的序列，该蛋白缺乏预测的质体转运肽，以及拟南芥 Arabidopsis thaliana 的两个溶血磷脂酸酰基转移酶 2（CaLPAT2a 和 CaLPAT2b）的同源物。CaKAS2B 在常规（WT）荠蓝和先前工程化的高油酸含量（HO）种子中的表达导致种子 VLCFA 含量增加。在 WT 和 HO 背景下的代表性转化体的 T3 种子中，总 VLCFA 水平分别从 24%提高到 35%，从 7%提高到 23%。在低内源细胞质脂肪酸延长活性的 HO 系中，岗桐酸是主要的 VLCFA，表明 CaKAS2B 延长到 C20 以外的能力有限。在荠蓝中表达 CaLPAT2b 导致种子 TAG 的 sn-2 位置的 C20-VLCFA 酯化显著增加，与相应对照系中的 sn-2 位置的 0.3%相比，一个转化系中的 VLCFA 水平为 33.8%。在转化系中观察到种子总 VLCFA 含量的微小变化，这意味着荠蓝中增加的 VLCFA 酯化能力导致 VLCFA 的位置再分配，但不会显著增加脂肪酸延长途径的通量。CaKAS2B 和 CaLPAT2a 用于工程化荠蓝中高岗桐酸水平的全部潜力仍有待确定。Consolida 和 Delphinium 的种子脂肪酸组成也为毛茛科的系统发育提供了有价值的信息。
