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

描述(申请人提供)：聚酮类天然产物是由多种生物体产生的结构复杂的分子，在医学上具有重要作用，最常用作抗生素和抗癌剂。由于目前使用的许多治疗方法的毒副作用，人们对开发新的化疗药物非常感兴趣；此外，由于抗生素耐药病原体(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。由于它们令人印象深刻的生物活性和通过化学合成获得多酮类化合物的挑战，人们在对产生这些天然产物的生物的生物合成途径进行基因重新编程方面进行了广泛的研究，并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以获得的生物活性化合物的多样性，必须对聚酮类化合物进行位置选择性修饰。一种使聚酮功能化的方法是在生物合成过程中对聚酮进行酶修饰(同时底物仍与酰基载体蛋白(ACP)结合)。酰基载体蛋白可以1)将底物直接传递给修饰酶，2)通过有利的蛋白质-蛋白质相互作用降低能量屏障，促进转化。聚酮类化合物的氧合作用对其生物活性有很大的影响，细胞色素P450单加氧酶在聚酮大环内酯类化合物的生物合成过程中经常起到定点选择性氧化作用。确实有 然而，一些报告表明，细胞色素P450酶作用于与ACP结合的底物。例如，BioI是一种细胞色素P450单加氧酶，参与生物素的生物合成，已知氧化C7/C8位的ACP结合的脂肪酸。我们建议使用BioI作为工程现场选择性C-H氧化聚酮的平台。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和增加氧化的kcat)。此外，我们将检查BioI的底物专一性，并确定ACP是否允许非天然底物经历氧化。最后，我们将把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，然后我们将与NAS和Debs一起共表达BioI，并确定BioI是否能够氧化聚酮合成酶生产线上产生的ACP结合的聚酮。我们将使用这些ACP-P450系统来设计聚酮的选择性C-H氧化，这将扩大通过生物合成获得的具有生物活性的大环内酯的多样性。

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.