Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum
Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum
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三角褐指藻新型硫酯酶 (PtTE) 的表征
DOI：
10.1002/jobm.201000520
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发表时间：
2011-12-01
影响因子：
3.1
通讯作者：
Jiang, Mulan
中科院分区：
文献类型：
--
作者：
Gong, Yangmin;Guo, Xiaojing;Jiang, Mulan
三角褐指藻（P. tricornutum）硫酯酶PtTE编码为648 bp的开放阅读框。216个氨基酸与植物酰基酰基载体蛋白（ACP）硫酯酶和细菌硫酯酶没有相似性。Southern blot分析显示，PtTE在三角蕨基因组中存在一个拷贝，Real-time定量PCR显示PtTE在氮剥夺后上调。通过在大肠杆菌XL1-Blue和脂肪酸β -氧化途径突变株K27fadD88中异源表达PtTE cDNA，建立了PtTE硫酯酶活性。通过重组菌株培养上清和膜脂的脂肪酸谱分析确定PtTE的底物特异性。重组PtTE使XL1-Blue的总脂肪酸含量提高了21%，并改变了膜脂和培养上清的脂肪酸组成。这些变化主要指向C18:0和C18:1脂肪酸。单独过表达PtTE不会改变三角虫的脂肪酸组成，但会使三角虫总脂肪酸含量增加72%。这种新型硫酯酶基因在提高微藻脂质产量的代谢工程中具有潜在的应用价值。这是迄今为止首次从真核微藻中发现硫酯酶。(c) 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
The Phaeodactylum tricornutum (P. tricornutum) thioesterase PtTE was encoded by a 648 bp open reading frame. The deduced 216 amino acids showed no similarity with plant acyl-acyl carrier protein (ACP) thioesterases and bacterial thioesterases. Southern blot analysis revealed that one copy of PtTE was present in the P. tricornutum genome, and Real-time quantitative PCR showed that PtTE was up-regulated upon nitrogen deprivation. Thioesterase activity of PtTE was established by heterologous expression of PtTE cDNA in Escherichia coli (E. coli) XL1-Blue and K27fadD88, a mutant strain of fatty acid beta-oxidation pathway. The substrate specificity of PtTE was determined by fatty acid profile analyses of the culture supernatant and membrane lipid of recombinant strains. Recombinant PtTE in E.coli enhanced total fatty acid content of XL1-Blue by 21%, and also changed the fatty acid compositions of membrane lipid and culture supernatant. These changes were directed predominantly towards C18:0 and C18:1 fatty acids. Overexpression of PtTE alone in P. tricornutum did not alter the fatty acid composition of P. tricornutum, but enhanced total fatty acid content by 72%. This novel thioesterase gene shows its potential in metabolic engineering for enhancing lipid yield of microalgae. This is so far the first report of thioesterase from eukaryotic microalgae. (c) 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)