Engineering Terpene Synthases for Increased Productivity

描述（由申请人提供）：萜烯是最多样化的一类天然产物，已知有30,000个成员。一些特征性萜形成商业上有用的产品，包括抗癌剂紫杉醇。大规模生产萜烯是困难的，因为目前的技术依赖于复杂的化学合成或低效的生物来源提取。为了满足对治疗用萜烯的需求，Keasling实验室对大肠杆菌进行了改造，使其能够生产抗疟疾化合物青蒿素的前体——阿莫菲-4,11-二烯。然而，萜烯合成酶，即摩phadiene synthase （ADS）的充分表达已成为增加摩phadiene产量的主要障碍。萜烯合成酶催化聚戊烯二磷酸环化形成萜烯主链，这是所有萜烯生物合成的第一步。在这篇文章中，我们概述了一种通过提高模型萜烯合成酶ADS的溶解度和活性来提高萜烯合成酶在大肠杆菌中的表达的方法，以及优化该酶的通量以增加amorphadiene的产量。将分析改变的ADS酶，并建立连接萜烯合成酶序列与酶功能的模型。

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.