Abstract

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and two-dimensional nuclear magnetic resonance to be 3-O-β-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-β-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol β-glucosides (β-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these β-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol β-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from UDP-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in total SG and almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of β-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

中文翻译：

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Aurantiochytrium limacinum mh0186中的C4-单甲基甾醇β-葡萄糖苷及其合酶

摘要

破囊壶菌，单细胞海洋原生生物，合成多不饱和脂肪酸 (PUFA) 和含 PUFA 的磷脂；然而，人们对它们的糖脂及其相关代谢知之甚少。在这里，我们报告了Aurantiochytrium limacinum mh0186中的两种糖脂 (GL-A、B) 及其合成酶。从A. limacinum中纯化出两种糖脂mh0186，经气相色谱、质谱和二维核磁共振测定为3-O-β-D-吡喃葡萄糖基-柱头-5,7,22-三烯（GL-A）和3-O- β-D-吡喃葡萄糖基-4α-甲基-柱头-7,22-二烯（GL-B），两者都是甾醇β-葡萄糖苷（β-SGs）；迄今为止，尚未报道 GL-B 的结构。使用酵母甾醇 β-葡糖基转移酶 (SGT; EC 2.4.1.173) 序列作为查询，从A. limacinum的基因组草图数据库中提取了负责合成这些 β-SG 的七个候选基因。使用酿酒酵母的表达分析揭示了两种基因产物（AlSGT-1和2）催化葡萄糖从UDP-葡萄糖转移到甾醇，产生甾基葡糖苷（SGs）。与 AlSGT-1 相比，AlSGT-2 对甾醇具有广泛的特异性，并使用 C4-单甲基甾醇合成 GL-B。在菌株 mh0186中alsgt-2而不是alsgt-1的破坏导致总 SG 减少和 GL-B 几乎完全丧失，表明 AlSGT-2 负责在A. limacinum mh0186 中合成 β-SG，尤其是具有独特甾醇结构的GL-B。