Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast Yarrowia lipolytica by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the SUR2 gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation. The Y. lipolytica SUR2 deletion (Ylsur2Δ) strain displayed retarded growth, increased pseudohyphal formation and stress sensitivity, along with the altered profiles of inositolphosphate-containing ceramides, GlcCers, and sterols. The subsequent disruption of the SLD1 gene, encoding a fungal/plant-specific Δ8 sphingolipid desaturase, restored filamentous growth in the Ylsur2Δ strain to a yeast-type form and further increased the production of human-type GlcCers. Additional introduction of mouse alkaline ceramidase 1 (maCER1) into the Ylsur2Δsld1Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the Ylsur2Δsld1Δ/maCER1 strain increased in proportion to the C/N ratio in the N-source optimized medium, leading to production of 1.4 g/L non-acetylated DHS at the 5 L fed-batch fermentation with glucose feeding. This study highlights the feasibility of using the engineered Y. lipolytica strains as a cell factory for valuable sphingolipid derivatives for pharmaceuticals, cosmeceuticals, and nutraceuticals.

中文翻译：

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解散耶罗菌中鞘脂生物合成途径的阐明和工程化，以增强人型鞘氨醇碱基和葡萄糖神经酰胺的产生


鞘脂是哺乳动物细胞、植物和各种微生物中的重要膜成分。我们旨在通过构建和表征具有特定基因缺失的突变菌株并整合外源基因来增强长链碱基 （LCB） 和葡萄糖神经酰胺 （GlcCers） 的产生，从而探索和开发油脂和二态酵母 Yarrowia lipolytica 中的鞘脂生物合成途径。为了阻断真菌/植物特异性植物鞘氨醇 （PHS） 途径，我们删除了编码鞘氨醇 C4-羟化酶的 SUR2 基因，导致二氢鞘氨醇 （DHS） 和鞘氨醇 （So） 的分泌产生显着升高，而没有乙酰化。Y. lipolytica SUR2 缺失 （Ylsur2Δ） 菌株表现出生长迟缓、假菌丝形成增加和应激敏感性，以及含肌醇磷酸神经酰胺、 GlcCers 和甾醇的谱改变。随后编码真菌/植物特异性 Δ8 鞘脂去饱和酶的 SLD1 基因被破坏，将 Ylsur2Δ 菌株中的丝状生长恢复到酵母型形式，并进一步增加了人型 GlcCers 的产生。将小鼠碱性神经酰胺酶 1 （maCER1） 额外引入 Ylsur2Δsld1Δ 双突变体中，显著增加了 DHS 和 So 的产生，同时降低了 GlcCers。在 N 源优化培养基中，Ylsur2Δsld1Δ/maCER1 菌株的 LCB 产量与 C/N 比率成比例增加，导致在 5 L 补料分批发酵和葡萄糖补料中产生 1.4 g/L 非乙酰化 DHS。本研究强调了使用工程化的 Y. lipolytica 菌株作为用于药物、药妆品和营养保健品的有价值的鞘脂衍生物的细胞工厂的可行性。