Engineering Terpene Synthases for Increased Productivity

描述（由申请人提供）：萜烯是最多样化的天然产物类别，已知有30，000种成员。许多具有特征的萜烯形成商业上有用的产品，包括抗癌剂紫杉醇。萜烯的大规模生产是困难的，因为目前的技术依赖于复杂的化学合成或从生物来源的低效提取。为了满足对治疗性萜烯的需求，Keasling实验室对大肠杆菌进行了改造，使其产生紫穗槐-4，11-二烯，这是抗疟疾化合物青蒿素的前体。然而，萜烯合酶紫穗槐二烯合酶（ADS）的充分表达已成为紫穗槐二烯产量增加的主要障碍。萜烯脱氢酶催化聚异戊二烯二磷酸环化形成萜烯骨架，这是所有萜烯生物合成中的第一个关键步骤。在这个建议中，我们概述了一种方法，以提高萜烯合酶表达在大肠杆菌。通过增加模型萜烯合酶ADS的溶解度和活性，以及优化通过该酶的通量以增加紫穗槐二烯的产量，来提高大肠杆菌的产量。将分析改变的ADS酶，并建立将萜烯合酶序列与酶功能联系起来的模型。

DESCRIPTION (provided by applicant): Terpenes are the most diverse class of natural products, with 30,000 known members. A number of characterized terpenes form commercially useful products, including the anticancer agent Taxol. Large- scale production of terpenes is difficult because current techniques rely on complex chemical synthesis or inefficient extraction from biological sources. To address the demand for therapeutic terpenes the Keasling laboratory has engineered Escherichia coli to produce amorpha-4,11-diene, the precursor for the anti- malarial compound, artemisinin. However, adequate expression of the terpene synthase, amorphadiene synthase (ADS), has become a major obstacle to increased amorphadiene production. Terpene synthases catalyze the cyclization of polyprenyl diphosphates to form a terpene backbone, which is the first committed step in all terpene biosynthesis. In this proposal, we outline an approach to improve terpene synthase expression in E. coli by increasing both solubility and activity,of the model terpene synthase, ADS, as well as to optimize flux through this enzyme to increase amorphadiene production. Altered ADS enzymes will be analyzed and a model linking terpene synthase sequence to enzyme function will be established.