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

描述（由申请人提供）：聚酮类天然产物是由多种生物产生的结构复杂的分子，在医学中具有重要作用，最常被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，开发新的化疗药物引起了极大的兴趣；此外，由于耐抗生素病原体（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聚酮类化合物的挑战，人们对产生这些天然产物的生物体的生物合成途径进行了广泛的基因重编程研究，并通过这种方法合成了许多类似物。为了进一步扩大可被合成生物学利用的生物活性化合物的多样性，必须开发聚酮类化合物的位点选择性修饰。一种功能化聚酮的方法是在生物合成过程中对聚酮进行酶修饰（而底物仍然与酰基载体蛋白（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-蛋白相互作用降低能量势垒，促进转化。多酮类化合物的氧化作用对其生物活性有显著影响，细胞色素P450单加氧酶通常在多酮类化合物生物合成后选择性氧化其大环内酯。有

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.