Identification, characterization, and expression of diacylgylcerol acyltransferase type-1 from Chlorella vulgaris
Identification, characterization, and expression of diacylgylcerol acyltransferase type-1 from Chlorella vulgaris
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DOI：
10.1016/j.algal.2015.10.017
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发表时间：
2016-01-01
影响因子：
5.1
通讯作者：
Lee-Parsons, Carolyn W. T.
中科院分区：
文献类型：
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作者：
Kirchner, Linda;Wirshing, Alison;Lee-Parsons, Carolyn W. T.
绿色微藻普通小球藻可以被诱导快速积累石油(干重40%)，并具有高饱和和单不饱和脂肪酸的生物柴油特征。在本研究中，我们测序了普通弯曲霉(CvuDGAT1)的二酰基甘油酰基转移酶-1(CvuDGAT1)，该酶催化三酰甘油(Tag)生物合成的最后一步，并利用生物信息学和酵母突变株的功能表达分析对该蛋白进行了鉴定。CvuDGAT1蛋白(460aa)具有其他植物和微藻DGAT1的特征基序，包括酰基辅酶A和二酰甘油底物的结合域以及可能的活性部位。此外，我们还报道了DGAT1的第一个假定的三级结构。该模型表明，尽管CvuDGAT1在蛋白质序列中广泛分离，但它整合到ER膜上，并预测底物结合结构域和活性部位的接近程度。我们还通过在一个不产油的酵母突变体中表达该基因并恢复石油水平，证明了CvuDGAT1的S在Tag生物合成中的功能。有趣的是，与莱茵衣藻不同，CvuDGAT1基因是高水平的结构性表达，可能有助于该物种积累高水平的石油(即40%的干重)。(C)2015爱思唯尔B.V.保留所有权利。
The green microalga Chlorella vulgaris can be induced to rapidly accumulate oil (40% by dry weight) with a biodiesel profile that is high in saturated and monounsaturated fatty acids. In this study, we sequenced the diacylglycerol acyltransferase type-1 from C. vulgaris (CvuDGAT1), which catalyzes the last step in triacylglycerol (TAG) biosynthesis, and characterized this protein using bioinformatics and functional expression assays in a yeast mutant. The CvuDGAT1 protein (460 aa) shares motifs characteristic of other plant and microalga DGAT1s, including the binding domains for the acyl-CoA and diacylglycerol substrates and the putative active site. In addition, we report the first putative tertiary structure of DGAT1. This model shows that CvuDGAT1 is integrated into the ER membrane and predicts the proximity of the substrate-binding domains and the active site, despite being widely separated in the protein sequence. We also demonstrate CvuDGAT1's function in TAG biosynthesis by expressing the gene and restoring oil levels in a non-oil-producing yeast mutant. Interestingly, unlike Chlamydomonas reinhardtii, the CvuDGAT1 gene is constitutively expressed at high levels and may contribute to this species' ability to accumulate high levels of oil (i.e. 40% by dry weight). (C) 2015 Elsevier B.V. All rights reserved.