C20- and C22-dihydroxy fatty acids (DiHFAs) are bioactive lipid mediators (LMs) in humans. Among them, leukotrienes are inflammatory mediators, whereas resolvins generated by M2 macrophages in humans are anti-inflammatory mediators. However, the synthesis of LMs by chemical or biological methods is inefficient. Here, we discovered a double-oxygenating arachidonate (ARA) 15R-lipoxygenase (15R-LOX) with the highest catalytic activity among the reported ARA LOXs. Cells expressing double-oxygenating 15R-LOX converted ARA, eicosapentaenoic acid, and docosahexaenoic acid (DHA) and DHA-rich fish oil hydrolyzates into 5R,15R-dihydroxyeicosatetraenoic acid, 5R,15R-dihydroxyeicosapentaenoic acid, and 7R,17R-dihydroxydocosahexaenoic acid as isomers of leukotriene B4, resolvin E4, and resolvin D5, which were identified as new compounds, within 1.5 h, with concentrations >1.5 g L⁻¹, conversions >77% (w/w), and productivity >1.0 g L⁻¹ h⁻¹, respectively. Double-oxygenating 15R-LOX was altered to single-oxygenating 15R-LOX by the L606F mutation using structure-guided engineering. Cells expressing single-oxygenating 15R-LOX produced 15R-hydroxyeicosatetraenoic acid, 15R-hydroxyeicosapentaenoic acid, and 17R-hydroxydocosahexaenoic acid, respectively. These results demonstrated that the six medicinally important LMs could be prepared from C20- and C22-polyunsaturated fatty acids and inexpensive DHA-rich fish oil hydrolyzates by cost-effective, eco-friendly, and efficient biosynthesis using the discovered double-oxygenating and engineered single-oxygenating LOXs.

中文翻译：

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使用双氧化 15R-脂氧合酶将 C20 和 C22 多不饱和脂肪酸和鱼油水解物生物转化为 R,R-二羟基脂肪酸作为脂质介质

C20- 和 C22- 二羟基脂肪酸 (DiHFA) 是人类生物活性脂质介质 (LM)。其中，白三烯是炎症介质，而人体M2巨噬细胞产生的分解素是抗炎介质。然而，通过化学或生物方法合成LMs效率低下。在这里，我们发现了一种双氧化花生四烯酸（ARA）15 R -脂氧合酶（15 R -LOX），在已报道的 ARA LOX 中具有最高的催化活性。表达双氧化 15 R -LOX 转化的 ARA、二十碳五烯酸和二十二碳六烯酸 (DHA) 以及富含 DHA 的鱼油水解物为 5 R ,15 R -二羟基二十碳四烯酸、5 R ,15 R -二羟基二十碳五烯酸和 7 R 的细胞,17 R -二羟基二十二碳六烯酸作为白三烯 B4、resolvin E4 和 resolvin D5 的异构体，在 1.5 小时内被鉴定为新化合物，浓度 >1.5 g L ^-1，转化率 >77% (w/w)，并且生产率分别>1.0 g L ^-1 h ^-1。使用结构引导工程通过 L606F 突变将双氧化 15 R -LOX 改变为单氧化 15 R -LOX。表达单氧化15R-LOX的细胞分别产生15R-羟基二十碳四烯酸、15R-羟基二十碳五烯酸和17R-羟基二十二碳六烯酸。这些结果表明，使用发现的双氧化和工程化的单链，可以通过经济高效、环境友好和高效的生物合成，从 C20 和 C22 多不饱和脂肪酸和廉价的富含 DHA 的鱼油水解物制备六种具有药用价值的 LM。 -氧化液氧。