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

• 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
• 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

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的毒副作用，对开发新的化学治疗剂存在极大的兴趣;另外，由于耐药性病原体（例如耐甲氧西林金黄色葡萄球菌，MRSA）的快速出现，迫切需要开发新的抗生素。由于它们令人印象深刻的生物活性和通过化学合成获得聚酮化合物的挑战，在对产生这些天然产物的生物体的生物合成途径进行遗传重编程方面已经进行了广泛的研究，并且已经通过这种方法合成了许多类似物。为了进一步扩大生物活性化合物的多样性，可以通过合成生物学，聚酮化合物的位点选择性修饰必须开发。使聚酮化合物官能化的一种方法是在生物合成过程中酶促修饰聚酮化合物（同时底物保持与酰基载体蛋白（ACP）结合）。酰基载体蛋白可以1）将底物直接递送至修饰酶，并且2）通过有利的蛋白质-蛋白质相互作用降低能量屏障来促进转化。 聚酮化合物的氧化可对其生物活性产生显著影响，并且细胞色素P450单加氧酶通常在聚酮化合物大环内酯的生物合成后起位点选择性氧化聚酮化合物大环内酯的作用。有 然而，有几个报道细胞色素P450酶作用于与ACP结合的底物。例如，BioI是参与生物素生物合成的细胞色素P450单加氧酶，已知其在C7/C8位置氧化ACP结合的脂肪酸。我们建议使用BioI作为一个平台的工程位点选择性C-H氧化聚酮化合物。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和增加氧化的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进行氧化。最后，我们将BioI整合到南昌霉素合成酶（NANS）和红霉素合成酶（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，然后我们将与NANS和DEBS共表达BioI沿着，并确定BioI是否能够氧化在聚酮化合物合成酶装配线上产生的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，这将扩大通过生物合成获得的生物活性大环内酯的多样性。