Oleaginous microalgae represent a valuable resource for the production of high-value molecules. Considering the importance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high-value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand; however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA. Recently, we have engineered the marine diatom Phaeodactylum tricornutum to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG, we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native P. tricornutum DGAT2B was taken forward for detailed characterisation. Overexpression of the endogenous P. tricornutum DGAT2B was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. PtDGAT2B has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous DGAT2B resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous DGAT2B and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment. This study provides further insight into lipid metabolism in P. tricornutum and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.

中文翻译：

---

海洋硅藻三角褐指藻中内源性 2 型二酰基甘油酰基转移酶的过度表达增强了脂质生成和 omega-3 长链多不饱和脂肪酸含量。

含油微藻代表了生产高价值分子的宝贵资源。考虑到 omega-3 长链多不饱和脂肪酸 (LC-PUFA) 对人类健康和营养的重要性，需要显着提高高价值二十碳五烯酸 (EPA) 和二十二碳六烯酸 (DHA) 的产量以满足需求；然而，目前的生产成本仍然很高。一种有前途的方法是对富含 EPA 和 DHA 的甘油三酯 (TAG) 水平提高的菌株进行代谢工程改造。最近，我们设计了海洋硅藻三角褐指藻以在 TAG 中积累更高水平的 DHA。为了进一步改善 omega-3 LC-PUFA 在 TAG 中的结合，我们将努力集中在 2 型酰基辅酶 A 的鉴定上：二酰基甘油酰基转移酶 (DGAT) 能够改善脂质生成和 DHA 在 TAG 中的掺入。DGAT是脂质合成中的关键酶。在对候选 DGAT 进行基于硅藻的体内筛选后，对原生三角褐指藻 DGAT2B 进行了详细表征。qRT-PCR 证实了内源性三角褐指藻 DGAT2B 的过表达，与野生型相比，转基因菌株生长成功。PtDGAT2B 具有广泛的底物特异性，偏好 C16 和 LC-PUFAs 酰基。此外，内源性 DGAT2B 的过表达导致更高的脂质产量和 TAG 中 DHA 水平的提高。此外，内源性 DGAT2B 的过表达和 Δ5-延长酶的异位表达的组合显示了如何使用迭代代谢工程来增加 DHA 和 TAG 含量，与氮处理无关。本研究进一步深入了解三角褐指藻中的脂质代谢，并提出了一种代谢工程方法，用于在微藻中高效生产 EPA 和 DHA。