Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi, Jiangsu Province, People's Republic of China.
Department of Clinical Laboratory, The Affiliated Wuxi People's Hospital of Nanjing Medical University, People's Republic of China.
Microbiol Spectr. 2023 Jun 15;11(3):e0013023. doi: 10.1128/spectrum.00130-23. Epub 2023 Apr 24.
Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in . This study provides an effective strain improvement method to enhance EPA accumulation in spp. , a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.
二十碳五烯酸（EPA）是一种对人体健康至关重要的ω-3 长链多不饱和脂肪酸（PUFA）。海洋真核生物是一种已被广泛用于合成 PUFA 的生物。目前，通过发酵生产 EPA 的效率低下，限制了其在 EPA 商业生产中的前景。由于 spp. 中 EPA 的合成途径仍不清楚，因此仍然需要诱变与高效筛选方法相结合。在这项研究中，基于大气压室温等离子体（ARTP）和硫酸二甲酯（DES）的两步渐进诱变方法，结合多次应激（肟草醚、三氯生和 2,2'-联吡啶）化合物筛选，开发了一种新的筛选策略。最终，从野生型（W）菌株中筛选出脂类（增加 1.57 倍，31.71 g/L）和 EPA（增加 5.64 倍，1.86 g/L）产量均增加的突变株 DBT-64；突变株 DBT-64 的二十二碳六烯酸（DHA）含量比野生型菌株低 11.41%。比较转录组分析表明，突变株中与聚酮合酶、脂肪酸延长和三酰基甘油合成途径相关的基因表达显著上调，而涉及β-氧化途径和脂肪酸降解途径的基因表达下调，有利于 spp. 中 EPA 的生物合成。这项研究为提高 spp. 中 EPA 的积累提供了一种有效的菌株改良方法，海洋真核微生物已成为多不饱和脂肪酸（PUFA）商业生产的候选生物。EPA 是一种ω-3 PUFA，对心血管疾病、精神分裂症等疾病具有预防和治疗作用。目前，通过 spp. 生产 EPA 的效率低下，限制了其商业化。在这项研究中，我们进行了基于 ARTP 和 DES 的两步渐进诱变，并筛选了多种应激（肟草醚、三氯生和 2,2'-联吡啶）以获得高 EPA 产量的突变体。此外，通过转录组分析证实了突变体中聚酮合酶途径、脂肪酸延长途径和三酰基甘油合成途径的高表达。因此，本研究建立的多应激筛选平台对 spp. 的 EPA 生产具有重要意义，并为通过基因工程手段调节微藻油脂中 EPA 的比例提供了有价值的信息。
