A New Approach for Engineering Polyketides by Cytochrome P450 C-H Oxidation

细胞色素 P450 C-H 氧化工程聚酮化合物的新方法

基本信息

批准号：
8526878
负责人：
Robert Vincent O'Brien
金额：
$ 4.92万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2016-06-26
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8526878
关键词：
Active Sites Acyl Carrier Protein Adverse effects Affect Anabolism Antibiotic Resistance Antibiotics Antineoplastic Agents Bacterial Infections Binding Biochemical Biological Biological Factors Biotin Carrier Proteins Complex Cytochrome P450 Disease Engineering Enzymes Erythromycin Fatty Acids Genetic Engineering Goals Kinetics Macrolides Malignant Neoplasms Medicine Mixed Function Oxygenases Modification Organism Pathway interactions Polyketide Macrolides Positioning Attribute Process Protein Binding Reaction Reporting Research Research Proposals Roentgen Rays Role Site Structure Substrate Specificity System analog chemical synthesis chemotherapeutic agent fatty acid oxidation interest methicillin resistant Staphylococcus aureus microorganism nanchangmycin novel strategies oxidation pathogen polyketide synthase programs protein protein interaction public health relevance synthetic biology tool

项目摘要

DESCRIPTION (provided by applicant): Polyketide natural products are structurally complex molecules produced by a variety of organisms and serve an important role in medicine, most often utilized as antibiotic and anticancer agents. There is great interest in developing new chemotherapeutic agents due to the toxic side effects of many currently utilized therapies; additionally, there is an urgent need to develop new antibiotics due to the rapid emergence of antibiotic-resistant pathogens (e.g. methicillin-resistant Staphylococcus aureus, MRSA). Because of their impressive biological activity and the challenge of accessing polyketides through chemical synthesis, there has been extensive research in genetically reprogramming the biosynthetic pathways of the organisms that produce these natural products, and numerous analogs have been synthesized by this approach. In order to further expand the diversity of biologically active compounds that can be accessed by synthetic biology, site-selective modification of polyketides must be developed. One approach to functionalize polyketides is to enzymatically modify the polyketide during the process of biosynthesis (while the substrate remains bound to an acyl carrier protein (ACP)). The acyl carrier protein could 1) deliver the substrate directly to the modifying enzyme, and 2) facilitate the transformation by lowering the energy barrier through favorable protein-protein interactions. Oxygenation of polyketides can have a dramatic impact on their biologically activity, and cytochrome P450 monooxygenases often act to site-selectively oxidize polyketide macrolides following their biosynthesis. There are several reports, however, of cytochrome P450 enzymes that act on substrates bound to an ACP. For example, BioI is a cytochrome P450 monooxygenase involved in biotin biosynthesis that is known to oxidize ACP-bound fatty acids at the C7/C8 positions. We propose to use BioI as a platform for engineering site-selective C-H oxidation of polyketides. To achieve this goal, we will study the Michaelis-Menten kinetics of BioI oxidation in order to determine whether the ACP facilitates the C-H oxidation (by lowering Km and increasing the kcat of the oxidation). In addition, we will examine the substrate specificity for BioI and determine whether the ACP will allow non-native substrates to undergo oxidation. Finally, we will incorporate BioI into module 2 of the nanchangmycin synthase (NANS) and erythromycin synthase (DEBS). We will first establish whether BioI is capable of oxidizing polyketides bound to isolated ACPs, and we will then co-express BioI along with NANS and DEBS, and determine whether BioI can oxidize ACP bound polyketides that are generated on a polyketide synthase assembly line. We will use these ACP-P450 systems to engineer site-selective C-H oxidation of polyketides, which will expand the diversity of biologically active macrolides accessible through biotic synthesis.

描述（由申请人提供）：聚酮化合物天然产物是由多种生物体产生的结构复杂的分子，在医学中发挥着重要作用，最常用作抗生素和抗癌剂。由于许多目前使用的疗法存在毒副作用，因此人们对开发新的化疗药物产生了极大的兴趣；此外，由于耐抗生素病原体（例如耐甲氧西林金黄色葡萄球菌，MRSA）的迅速出现，迫切需要开发新的抗生素。由于其令人印象深刻的生物活性以及通过化学合成获得聚酮化合物的挑战，人们在对产生这些天然产物的生物体的生物合成途径进行基因重编程方面进行了广泛的研究，并且通过这种方法合成了许多类似物。为了进一步扩大合成生物学可获取的生物活性化合物的多样性，必须开发聚酮化合物的位点选择性修饰。功能化聚酮化合物的一种方法是在生物合成过程中对聚酮化合物进行酶促修饰（同时底物仍与酰基载体蛋白（ACP）结合）。酰基载体蛋白可以1）将底物直接递送至修饰酶，2）通过有利的蛋白质-蛋白质相互作用降低能量势垒来促进转化。聚酮化合物的氧化可对其生物活性产生巨大影响，并且细胞色素 P450 单加氧酶通常在生物合成后选择性地氧化聚酮化合物大环内酯。有然而，有几份报告表明细胞色素 P450 酶作用于与 ACP 结合的底物。例如，BioI 是一种参与生物素生物合成的细胞色素 P450 单加氧酶，已知它可以氧化 C7/C8 位置上的 ACP 结合脂肪酸。我们建议使用 BioI 作为工程聚酮化合物的位点选择性 C-H 氧化的平台。为了实现这一目标，我们将研究 BioI 氧化的 Michaelis-Menten 动力学，以确定 ACP 是否促进 C-H 氧化（通过降低 Km 并增加氧化的 kcat）。此外，我们将检查 BioI 的底物特异性，并确定 ACP 是否允许非天然底物发生氧化。最后，我们将 BioI 合并到南昌霉素合酶 (NANS) 和红霉素合酶 (DEBS) 的模块 2 中。我们将首先确定 BioI 是否能够氧化与分离的 ACP 结合的聚酮化合物，然后我们将 BioI 与 NANS 和 DEBS 共表达，并确定 BioI 是否可以氧化在聚酮化合物合酶装配线上生成的 ACP 结合的聚酮化合物。我们将使用这些 ACP-P450 系统来设计聚酮化合物的位点选择性 C-H 氧化，这将扩大可通过生物合成获得的生物活性大环内酯的多样性。