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首页> 外文期刊>Applied Microbiology >Type II Diacylglycerol Acyltransferase from Claviceps purpurea with Ricinoleic Acid, a Hydroxyl Fatty Acid of Industrial Importance, as Preferred Substrate
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Type II Diacylglycerol Acyltransferase from Claviceps purpurea with Ricinoleic Acid, a Hydroxyl Fatty Acid of Industrial Importance, as Preferred Substrate

机译：优选的底物为紫杉酸（Claviceps purpurea）的II型二酰基甘油酰基转移酶，以蓖麻油酸（一种具有工业重要性的羟基脂肪酸）为优选底物

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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. .
机译：引起谷物性麦角麦芽病的真菌病原体-紫薇（Claviceps purpurea）产生的甘油酯在其菌核中含有高含量的蓖麻油酸[（R）-12-羟基十八烷基-顺式-9-烯酸]。最近，从该真菌中鉴定了涉及蓖麻油酸的生物合成的脂肪酸羟化酶（C.purpurea FAH [CpFAH]）（D.Meesapyodsuk和X.Qiu，Plant Physiol.147.1325-1333，2008）。在这里，我们描述了参与蓖麻油酸到甘油三酸酯组装过程中的紫杉II型二酰基甘油酰基转移酶（CpDGAT2）的克隆和生化特性。通过简并RT-PCR（逆转录PCR）克隆CpDGAT2基因。该基因的表达恢复了四重突变酿酒酵母H1246中三酰基甘油（TAG）的体内合成，其中所有四个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比亚油酸，油酸或亚麻酸更优选蓖麻油酸作为酰基供体，相对于1,2-，CpDGAT2更优选1,2-油酰-sn-甘油二棕榈酰-sn-甘油作为酰基受体。与CpFAH与天然酵母DGAT2（酿酒酵母DGA1 [ScDGA1]）的共表达或单独CpFAH的表达相比，酵母中CpFAH与CpDGAT2的共表达导致蓖麻油酸的积累增加。 Northern印迹分析表明CpFAH仅在菌核细胞中表达，而在菌丝体或分生孢子细胞中未检测到该基因的转录本。尽管在分生孢子中表达很低，但CpDGAT2在所研究的细胞类型中表达更为广泛。 CpDGAT2和CpFAH在菌核细胞中的高表达，其中高水平的蓖麻油酸酯甘油酯积聚，提供了进一步的证据，支持CpDGAT2和CpFAH作为紫花梭菌中蓖麻油酸合成和组装的关键酶的作用。 。

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