As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear. In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain. Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

中文翻译：

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聚酮合酶基因簇的 ORFC 对Schizochytrium limacinum SR21脂肪酸积累的作用

作为多不饱和脂肪酸 (PUFA) 的潜在来源，裂殖壶菌属。已广泛应用于工业生产PUFA。聚酮合酶 (PKS) 簇被认为是裂殖壶菌属中 PUFA 合成的主要途径。作为 PKS 簇中的三个开放阅读框 (ORF) 之一，ORF 在脂肪酸生物合成中起着至关重要的作用。然而，ORFC 域在裂殖壶菌属脂肪酸合成中的功能。仍然不清楚。在这项研究中，进行了异源表达和过度表达以研究 ORFC 及其结构域在脂肪酸积累中的作用。首先，ORFC在酵母中异源表达，显着增加了PUFA含量。然后，位于 ORF 上的脱水酶 (DH) 和烯酰还原酶 (ER) 域分别在 Schizochytrium limacinum SR21 中过表达。脂肪酸谱分析表明，DH 和 ER 过表达菌株中 PUFA 和饱和脂肪酸的含量分别增加。这表明 DH 和 ER 结构域在脂质积累中起着不同的作用。代谢和转录组学分析表明，在 DH 过表达菌株中，磷酸戊糖途径和三酰甘油生物合成增强，而三羧酸循环和脂肪酸氧化减弱。然而，在 ER 过表达菌株中发现了相反的效果。因此，脂肪酸的生物合成需要ORF。DH 结构域在 PUFA 合成中起关键作用，而 ER 结构域可能与 Schizochytrium limacinum SR21 中的饱和脂肪酸 (SFA) 合成有关。