Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Appl Environ Microbiol. 2010 Feb;76(4):1135-42. doi: 10.1128/AEM.02297-09. Epub 2009 Dec 18.
Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea.
麦角菌是一种引起谷类作物麦角病的真菌病原体，它在菌核中产生含有高水平蓖麻酸[(R)-12-羟基十八碳顺-9-烯酸]的甘油酯。最近，从这种真菌中鉴定出一种参与蓖麻酸生物合成的脂肪酸羟化酶（C. purpurea FAH [CpFAH]）（D. Meesapyodsuk 和 X. Qiu，植物生理学。147:1325-1333，2008）。在这里，我们描述了参与蓖麻酸组装成三酰基甘油的麦角菌 II 型二酰基甘油酰基转移酶（CpDGAT2）的克隆和生化特性。CpDGAT2 基因是通过简并 RT-PCR（反转录-PCR）克隆的。该基因的表达恢复了四重突变株酿酒酵母 H1246 中体内三酰基甘油（TAG）的合成，该突变株中所有四个 TAG 生物合成基因（DGA1、LRO1、ARE1 和 ARE2）均被破坏。使用转化酵母菌株的微粒体制剂进行的体外酶促测定表明，CpDGAT2 优先选择蓖麻酸作为酰基供体，而不是亚油酸、油酸或亚麻酸，并且它优先选择 1,2-二油酰基-sn-甘油作为酰基受体，而不是 1,2-二棕榈酰基-sn-甘油。CpFAH 与 CpDGAT2 在酵母中的共表达导致与 CpFAH 与天然酵母 DGAT2（S. cerevisiae DGA1 [ScDGA1]）或 CpFAH 单独表达的共表达相比，蓖麻酸的积累增加。Northern blot 分析表明，CpFAH 仅在菌核细胞中表达，在菌丝或分生孢子细胞中未检测到该基因的转录物。CpDGAT2 在检查的细胞类型中表达更为广泛，尽管在分生孢子中表达水平较低。CpDGAT2 和 CpFAH 在积累高含量蓖麻酸的菌核细胞中的高表达进一步证明了它们作为麦角菌中蓖麻酸合成和组装的关键酶的作用。
