Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway
Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway
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DOI：
10.1073/pnas.1000532107
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发表时间：
2010-06-22
影响因子：
11.1
通讯作者：
Kuzuyama, Tomohisa
中科院分区：
文献类型：
--
作者：
Okamura, Eiji;Tomita, Takeo;Kuzuyama, Tomohisa
乙酰乙酰辅酶A是甲羟戊酸途径中3-羟基-3-甲基戊二酰（HMG）辅酶A的前体，其对于萜类骨架生物合成是必需的。乙酰乙酰辅酶A也是聚-β-羟基丁酸酯的前体，聚-β-羟基丁酸酯是一种由微生物产生的聚酯类聚合物。乙酰乙酰辅酶A的从头合成通常由乙酰乙酰辅酶A硫解酶通过两个乙酰辅酶A分子之间的硫酯依赖性克莱森缩合反应催化。在这里，我们报告nphT 7，发现在甲羟戊酸途径基因簇从土壤分离的链霉菌属菌株，编码一种不寻常的乙酰乙酰辅酶A合成酶。在大肠杆菌中过表达的重组酶催化乙酰辅酶A和丙二酰辅酶A的单一缩合，得到乙酰乙酰辅酶A和辅酶A。用丙二酰-（酰基载体蛋白）替换丙二酰-CoA导致缩合活性的损失。通过两分子乙酰辅酶A的缩合，未检测到乙酰乙酰辅酶A合成活性。基于NphT 7的这些特性，我们建议将这种不寻常的硫解酶超家族乙酰乙酰辅酶A合酶命名为。在异源宿主中共表达nphT 7与HMG-CoA合酶基因和HMG-CoA还原酶基因允许甲羟戊酸的产量比仅表达HMG-CoA合酶和HMG-CoA还原酶基因时高3.5倍。该结果表明nphT 7可用于显著增加细胞中乙酰乙酰辅酶A的浓度，最终导致有用的萜类化合物和聚-β-羟基丁酸酯的产生。
Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-beta-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen condensation reaction between two molecules of acetyl-CoA. Here, we report that nphT7, found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain, encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acetyl-CoA. Based on these properties of NphT7, we propose to name this unusual enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Co-expression of nphT7 with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase and HMG-CoA reductase genes were expressed. This result suggests that nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-beta-hydroxybutyrate.