Biochemical and Structural Studies of A Novel Simvastatin Synthase

描述(申请人提供)：天然产品是农业和医药应用的生物活性化合物的极其重要的来源。参与次生代谢的酶作为生物催化剂具有很大的潜力，可用于精细化学品和高价值药物的高效合成。在代谢工程小组和结构生物学小组之间的合作工作中，我们将收获这一潜力，使其能够一步合成重磅炸弹药物辛伐他汀(Zocor.)。目前，辛伐他汀是由天然产物洛伐他汀通过低效的多步工艺合成的。我们建议的辛伐他汀生物合成将产生一种全新的工艺，可以成为当前化学路线之外的一种有吸引力的替代方案。这项研究的中心酶是洛伐他汀生物合成途径中的一种酰基转移酶LovD。我们已经对该酶进行了广泛的、初步的生化表征，以表明LovD是一种辛伐他汀合成酶，并有可能被工程化为辛伐他汀生物合成的强大生物催化剂。这项建议将审查以下具体目标： 目的1：LovD的定向进化。我们将使用定向进化方法来提高LovD对辛伐他汀合成的催化效率。我们建立了一种基于辛伐他汀形成的高通量筛选方法。 目标2：基于结构的洛夫丁工程。洛夫德的X射线晶体结构将在叶芝实验室进行研究。对结构分析确定的关键残基进行合理的突变，以探索LovD的功能，并改善LovD对辛伐他汀合成的催化性能。 目的3：大肠杆菌作为全细胞生物催化剂的代谢工程。我们将改造大肠杆菌的多药物转运系统，以提高其向细胞外空间输出辛伐他汀的效率。这将提高整个电池反应器在高产品浓度下的转化率。 目的4：土曲霉直接生物合成辛伐他汀。我们将通过代谢工程使A.terreus在洛伐他汀的生物合成中受阻，但在辛伐他汀的生物合成中受到抑制。项目叙事 我们建议进行生化和结构研究，以研究最近从我们的实验室发现的一种辛伐他汀合成酶。我们将利用蛋白质和代谢工程方法开发一种能够生物合成辛伐他汀的全细胞生物催化剂。这项工作将是生物催化的一个重要里程碑，但将酶应用于合成具有重要商业意义的化合物如辛伐他汀的报道尚未见报道。将天然产物生物合成酶设计成有用的生物催化剂的成功结果可能会导致从生物催化的角度研究这类迷人的酶的额外努力。同时，拟议工作的结果将为蛋白质工程、酶结构和功能、大肠杆菌膜运输和曲霉代谢提供重要的科学见解。

DESCRIPTION (provided by applicant): Natural products are extremely important sources of bioactive compounds for agricultural and pharmaceutical applications. Enzymes involved in secondary metabolism hold great potential as biocatalysts that may be used in the efficient synthesis of fine chemicals and high value pharmaceuticals. In this collaborative work between a metabolic engineering group and a structural biology group, we will harvest this potential towards the one-step synthesis of the blockbuster drug simvastatin (Zocor.). Simvastatin is currently synthesized from the natural product lovastatin via inefficient, multistep processes. Our proposed biosynthesis of simvastatin will result in a completely novel process that can be an attractive alternative over the current chemical routes. The central enzyme in this study is LovD, an acyltransferase from the lovastatin biosynthetic pathway. We have performed extensive, preliminary biochemical characterization of this enzyme to show that LovD is a simvastatin synthase, and can be potentially engineered into a powerful biocatalyst for simvastatin biosynthesis. This proposal will examine the following specific aims: AIM 1: Directed Evolution of LovD. We will use directed evolution methods to improve the catalytic efficiencies of LovD towards simvastatin synthesis. We have developed a high throughput screening assay based on the formation of simvastatin. AIM 2: Structure-Based Engineering of LovD. The X-ray crystal structure of LovD will be pursued in the Yeates Lab. Rational mutagenesis of key residues identified from structural analysis will be performed to probe LovD function and improve LovD catalytic properties towards simvastatin synthesis. AIM 3: Metabolic Engineering of E. coli as a whole cell biocatalyst. We will engineer the multidrug transporter system of E. coli to improve its efficiency in exporting simvastatin to the extracellular space. This will improve the conversion of the whole cell reactor at high product concentrations. AIM 4: Direct Biosynthesis of Simvastatin from A. terreus. We will metabolically engineer A. terreus to be blocked in lovastatin biosynthesis, but robust in simvastatin biosynthesis. Project Narrative We have proposed biochemical and structural studies to investigate a simvastatin synthase recently identified from our laboratories. We will use protein and metabolic engineering methods to develop a whole cell biocatalyst that can biosynthesize simvastatin. This work will represent an important milestone in biocatalysis, application of enzymes towards the synthesis of a compound as commercially important as simvastatin has not been reported. The successful outcome of engineering a natural product biosynthetic enzyme into a useful biocatalyst may lead to additional efforts to examine this class of fascinating enzymes from a biocatalysis prospective. At the same time, the outcome of the proposed work will provide important scientific insight into protein engineering, enzyme structure and function, E. coli membrane transport, and Aspergillus metabolism.