Very long-chain polyunsaturated fatty acids (VLCPUFAs) are essential components of cell membranes and precursors for bioactive compounds regulating important physiological processes in humans and animals. Lack or imbalance of these fatty acids can lead to various physiological problems in humans such as immunological disorders, neurological conditions and cardiovascular diseases (Bazinet and Laye, 2014). The current market and transgenic plant production of VLCPUFAs is primarily focused on two ω3 VLCPUFAs, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3), while other VLCPUFAs have been overlooked (Ganesh and Hettiarachchy, 2016; Napier et al., 2019; Qiu et al., 2020). Docosatrienoic acid (DTA, 22:3n-3) is an ω3 VLCPUFA with 22 carbons and three double bonds at 13, 16 and 19 positions, and was recently found to possess anti-inflammatory and antitumor properties comparable to DHA with potential nutraceutical and cosmetic uses (Chen et al., 2021). Biosynthesis of DTA follows the elongation and desaturation pathways of ω6 and ω3 polyunsaturated fatty acids (PUFAs; Figure 1a). In the ω3 pathway, α-linolenic acid (ALA, 18:3n-3) is elongated to eicosatrienoic acid (ETA, 20:3n-3) which is then elongated again to DTA (22:3n-3) by a single ELO type elongase (EhELO1) (Meesapyodsuk et al., 2018). In the ω6 pathway, linoleic acid (LA, 18:2n-6) is elongated to eicosadienoic acid (EDA, 20:2n-6) and elongated again to docosadienoic acid (DDA, 22:2n-6) by the same elongase. In addition, LA can be desaturated to ALA by a 18C-PUFA ω3 desaturase (CpDesX) while EDA can also be desaturated to ETA by a VLCPUFA ω3 desaturase (PiO3). Both desaturated products, ALA and ETA, can then be elongated to DTA by EhELO1.

To produce DTA in oilseed crop B. carinata, a dedicated oilseed crop in Canada for specialty oil, four constructs were made expressing the elongase and desaturases following the biosynthetic pathway (Figure 1b). Besides the expression cassettes with genes in the biosynthetic pathway, all constructs carried a herbicide phosphinothricin resistant gene under the control of a constitutive promoter for transformant selection (Meesapyodsuk et al., 2018). The first construct (Bc-1) expresses a single elongase EhELO1 from plant Eranthis hyemalis that can elongate a wide range of PUFAs, which has been reported previously (Meesapyodsuk et al., 2018). The second construct (Bc-2) expresses two genes encoding EhELO1 and CpDesX. CpDesX is an ω3 desaturase from fungus Claviceps purpurea with regioselectivity towards ω6-18C-PUFAs that can effectively convert LA to ALA (Meesapyodsuk et al., 2007). The third construct (Bc-3) expresses three genes coding for EhELO1, CpDesX and EhLPAAT2. EhLPAAT2 is an endoplasmic lysophosphatidic acid acyltransferase from E. hyemalis that can incorporate VLCPUFAs into the sn-2 position of triacylglycerols, as B. carinata lacks the capacity (Meesapyodsuk et al., 2021). The fourth construct (Bc-4) expresses EhELO1, CpDesX, EhLPAAT2 and PiO3. PiO3 is another ω3 desaturase from fungus Pythium irregulare that can convert ω6-VLCPUFAs to ω3-VLCPUFAs particularly for ω6-20C-VLCPUFAs (Cheng et al., 2010). Each of these genes in the four constructs was under the control of a seed-specific promoter, napin or conlinin, and an octopine synthase (OCS) terminator.

The three new constructs were introduced into a low erucic acid breeding line through a Agrobacterium-mediated transformation approach using petioles as explants (Cheng et al., 2010). Transgenic plants selected with phosphinothricin and genomic DNA PCR were grown in growth cabinets at 22 °C under a 16-h-light (120 μEm⁻²/s)/8-h-dark photoperiod. Fatty acid analysis of transgenic B. carinata by GC-FID expressing the first construct Bc-1 with EhELO1 alone has been reported previously (Meesapyodsuk et al., 2018), producing several new VLCPUFAs such as EDA, ETA, DDA and DTA. Among them, DDA was the most abundant, followed by EDA, DTA and ETA. Transgenic B. carinata expressing the second construct Bc-2 with two genes EhELO1 and CpDesX produced a similar fatty acid profile as Bc-1; however, the abundance of fatty acids varied significantly. Particularly, the abundance of LA and ALA was the reverse of each other in Bc-1 and Bc-2. This was due to the desaturation activity of CpDesX on LA, giving rise to ALA. The high level of ALA for elongation by EhELO1 resulted in the higher level of DTA than DDA in Bc-2 transgenic plants. Transgenic B. carinata expressing the third construct Bc-3 produced a similar fatty acid profile as Bc-2, but the amount of DTA was further increased, which was due to the pulling activity of EhLPAAT2 in the incorporation of VLCPUFAs into the sn-2 position of TAGs (Meesapyodsuk et al., 2021). Transgenic plants expressing the four gene construct Bc-4 produced a similar fatty acid profile as Bc-3, but with a higher level of ETA than EDA due to the desaturation from EDA to ETA catalysed by PiO3. The higher level of ETA for elongation by EhELO1 resulted in a further increase of DTA in Bc-4 (Figure 1c, Table S1). One elite line of Bc-4 transgenic plants was selected for propagation to the next generations. Fatty acid analysis of transgenic seeds from the three generations showed that the amount of DTA was slightly increased over these generations. DTA was in a range from 16% to 20% along with DDA in a range of 4%–6% over the three generations. The amount of DTA was more than three times that of DDA, accounting for 20% on average in the T3 transgenic plants (Table S2).

MALDI-TOF/MS was then utilized to profile the TAG species (Hong et al., 2002) in the seeds of T3 transgenic plants of selected elite lines with a single EhELO1 and four genes (EhELO1+CpDesX+EhLPAAT2+PiO3). Untransformed B. carinata produced three major fatty acids, 18:1 (oleic acid), LA and ALA where the major molecular species of TAGs were ALA/LA/18:1, LA/LA/LA, LA/LA/18:1, ALA/LA/LA and LA/18:1/18:1 according to the relative abundance. Transgenic B. carinata expressing EhELO1 alone produced many new TAG species such as DDA/ALA/EDA (or DTA/LA/EDA), DDA/18:1/18:1 (or 20:1/LA/20:1), DDA/LA/DDA (or DDA/18:1/DTA), and DDA/18:1/16:0 (or 20:1/LA/18:0). Those were also among the most abundant TAG species. As compared to those in wild type, molecular weights of TAGs in the EhELO1 transgenics were shifted up by 2 to 8 carbons, indicating one to two VLCPUFAs such as DDA, DTA and EDA were incorporated into TAGs. Transgenic B. carinata expressing the four genes (EhELO1+CpDesX+EhLPAAT2+PiO3) further produced many new TAG species with three VLCPUFAs such as DTA/DTA/24:3 and DTA/DTA/DDA. Among all TAG profiles produced, those TAG species with three VLCPUFAs were the most abundant, followed by those with one or two VLCPUFAs such as DTA/DDA/ALA, DTA/ALA/ETA, DTA/18:1/LA, DDA/ALA/16:0. In TAGs with one or two VLCPUFAs, the shift of the molecular weights down by 2 to 4 daltons, as compared to those in EhELO1 transgenic seeds, indicated an addition of one or two double bonds in acylated fatty acids (Figure S1).

In summary, ω3-VLCPUFA DTA possesses potential health benefits, but the source for this fatty acid does not exist in nature. This study employed a stepwise strategy to engineer the biosynthetic pathway in B. carinata; about 20% DTA of the total fatty acids was produced in the transgenics. No obvious phenotypic changes such as seed germination, seedling growth and development were observed in the plants. The amount of DTA in seeds remained stable over three generations where DTA was mainly placed in TAG species with two or three VLCPUFAs. Efficient and sustainable production of DTA in the oilseed crop provides an opportunity for this fatty acid to be used in cosmetics, foods and feeds.

极长链多不饱和脂肪酸 (VLCPUFA) 是细胞膜的重要组成部分，也是调节人类和动物重要生理过程的生物活性化合物的前体。这些脂肪酸的缺乏或失衡会导致人类出现各种生理问题，例如免疫系统疾病、神经系统疾病和心血管疾病（Bazinet 和 Laye，  2014 年）。目前VLCPUFAs的市场和转基因植物生产主要集中在两种ω3 VLCPUFAs，二十二碳六烯酸（DHA，22:6n-3），二十碳五烯酸（EPA，20:5n-3），而其他VLCPUFAs被忽视（Ganesh和Hettiarachchy，  2016 年；Napier等人，  2019 年；Qiu等人，  2020 年). 二十二碳三烯酸 (DTA, 22:3n-3) 是一种 ω3 VLCPUFA，在 13、16 和 19 位具有 22 个碳原子和三个双键，最近被发现具有与 DHA 相当的抗炎和抗肿瘤特性，具有潜在的营养保健品和化妆品用途使用（Chen等人，  2021 年）。DTA 的生物合成遵循 ω6 和 ω3 多不饱和脂肪酸（PUFA；图 1a）的延伸和去饱和途径。在 ω3 途径中，α-亚麻酸 (ALA, 18:3n-3) 被延长为二十碳三烯酸 (ETA, 20:3n-3)，然后通过单个 ELO 再次延长为 DTA (22:3n-3)型延伸酶 (EhELO1) (Meesapyodsuk et al .,  2018). 在 ω6 途径中，亚油酸 (LA, 18:2n-6) 被延长为二十碳二烯酸 (EDA, 20:2n-6)，并通过相同的延长酶再次延长为二十二碳二烯酸 (DDA, 22:2n-6)。此外，LA 可以通过 18C-PUFA ω3 去饱和酶 (CpDesX) 去饱和为 ALA，而 EDA 也可以通过 VLCPUFA ω3 去饱和酶 (PiO3) 去饱和为 ETA。然后，EhELO1 可以将两种去饱和产物 ALA 和 ETA 延长为 DTA。

为了在油籽作物B. carinata中生产 DTA ，加拿大一种专门用于生产特种油的油料作物，制备了四种表达生物合成途径后的延长酶和去饱和酶的构建体（图 1b）。除了在生物合成途径中具有基因的表达盒外，所有构建体都携带在组成型启动子控制下的除草剂膦丝菌素抗性基因，用于转化体选择（Meesapyodsuk等人，  2018 年）。第一个构建体 (Bc-1) 表达来自植物Eranthis hyemalis的单一延伸酶 EhELO1，它可以延伸广泛的 PUFA，这在之前已有报道（Meesapyodsuk等人，  2018 年）). 第二个构建体 (Bc-2) 表达两个编码 EhELO1 和 CpDesX 的基因。CpDesX 是一种来自真菌Claviceps purpurea的 ω3 去饱和酶，对 ω6-18C-PUFA 具有区域选择性，可以有效地将 LA 转化为 ALA（Meesapyodsuk等人，  2007 年）。第三个构建体 (Bc-3) 表达编码 EhELO1、CpDesX 和 EhLPAAT2 的三个基因。EhLPAAT2 是一种来自E. hyemalis的内质溶血磷脂酸酰基转移酶，它可以将 VLCPUFAs 整合到甘油三酯的sn-2位置，因为B. carinata缺乏这种能力（Meesapyodsuk等人，  2021 年）). 第四个构建体 (Bc-4) 表达 EhELO1、CpDesX、EhLPAAT2 和 PiO3。PiO3 是另一种来自真菌不规则腐霉的 ω3 去饱和酶，它可以将 ω6-VLCPUFA 转化为 ω3-VLCPUFA，尤其是 ω6-20C-VLCPUFA（Cheng等人，  2010 年）。四个构建体中的每个基因都在种子特异性启动子napin或conlinin和章鱼碱合酶 (OCS) 终止子的控制下。

这三种新构建体通过使用叶柄作为外植体的农杆菌介导的转化方法被引入到低芥酸育种系中（Cheng等人，  2010）。用草胺膦和基因组 DNA PCR 选择的转基因植物在 22 °C 的生长箱中在 16 小时光照 (120 μEm ^-2 /s)/8 小时黑暗光周期下生长。先前已经报道了通过 GC-FID 表达第一个构建体 Bc-1 和单独的EhELO1对转基因B. carinata进行脂肪酸分析（Meesapyodsuk等人，  2018 年），产生了几种新的 VLCPUFA，例如 EDA、ETA、DDA 和 DTA。其中，DDA含量最多，其次是EDA、DTA和ETA。转基因B. carinata表达具有两个基因EhELO1和CpDesX的第二个构建体 Bc-2产生与 Bc-1 相似的脂肪酸谱；然而，脂肪酸的丰度差异很大。特别地，LA和ALA的丰度在Bc-1和Bc-2中彼此相反。这是由于 CpDesX 对 LA 的去饱和活性，从而产生了 ALA。用于 EhELO1 延伸的高水平 ALA 导致 Bc-2 转基因植物中的 DTA 水平高于 DDA。表达第三个构建体 Bc-3 的转基因B. carinata产生与 Bc-2 相似的脂肪酸谱，但 DTA 的量进一步增加，这是由于 EhLPAAT2 在将 VLCPUFAs 掺入sn-2中的拉动活性TAG 的位置 (Meesapyodsuk et al .,  2021). 表达四种基因构建体 Bc-4 的转基因植物产生与 Bc-3 相似的脂肪酸谱，但由于 PiO3 催化 EDA 去饱和为 ETA，因此 ETA 水平高于 EDA。EhELO1 延伸的更高水平的 ETA 导致 Bc-4 中的 DTA 进一步增加（图 1c，表 S1）。选择 Bc-4 转基因植物的一个优良品系用于繁殖下一代。对三代转基因种子的脂肪酸分析表明，DTA 的量在这几代中略有增加。在三代中，DTA 在 16% 到 20% 的范围内，DDA 在 4% 到 6% 的范围内。DTA 的量是 DDA 的三倍多，在 T3 转基因植物中平均占 20%（表 S2）。

然后使用 MALDI-TOF/MS 来分析具有单个EhELO1和四个基因 ( EhELO1+CpDesX+EhLPAAT2+PiO3 ) 的选定优良品系的 T3 转基因植物种子中的 TAG 种类（Hong等人，  2002）。未转化的B. carinata产生三种主要脂肪酸，18:1（油酸）、LA 和 ALA，其中 TAG 的主要分子种类为 ALA/LA/18:1、LA/LA/LA、LA/LA/18:1 , ALA/LA/LA 和 LA/18:1/18:1 根据相对丰度。转基因B. carinata单独表达 EhELO1 产生了许多新的 TAG 种类，如 DDA/ALA/EDA（或 DTA/LA/EDA）、DDA/18:1/18:1（或 20:1/LA/20:1）、DDA/LA/ DDA（或 DDA/18:1/DTA）和 DDA/18:1/16:0（或 20:1/LA/18:0）。这些也是最丰富的 TAG 物种之一。与野生型相比，EhELO1转基因中 TAG 的分子量向上移动了 2 到 8 个碳，表明一到两个 VLCPUFA，如 DDA、DTA 和 EDA 被并入 TAG 中。转基因B. carinata表达这四个基因（EhELO1+CpDesX+EhLPAAT2+PiO3）进一步产生了许多具有三个VLCPUFAs的新TAG物种，如DTA/DTA/24:3和DTA/DTA/DDA。在产生的所有 TAG 图谱中，具有三个 VLCPUFA 的 TAG 种类最多，其次是具有一个或两个 VLCPUFA 的种类，如 DTA/DDA/ALA、DTA/ALA/ETA、DTA/18:1/LA、DDA/ALA /16:0。在具有一个或两个 VLCPUFA 的 TAG 中，与 EhELO1 转基因种子相比，分子量降低了 2 至 4 道尔顿，表明在酰化脂肪酸中添加了一个或两个双键（图 S1）。

总之，ω3-VLCPUFA DTA 具有潜在的健康益处，但这种脂肪酸的来源在自然界中并不存在。本研究采用逐步策略来设计B. carinata的生物合成途径；在转基因中产生了总脂肪酸的约20％的DTA。植株未见种子萌发、幼苗生长发育等明显表型变化。种子中 DTA 的量在三个世代内保持稳定，其中 DTA 主要放置在具有两个或三个 VLCPUFA 的 TAG 物种中。在油籽作物中高效且可持续地生产 DTA 为这种脂肪酸用于化妆品、食品和饲料提供了机会。