Engineered Biosynthesis of Novel Macrolactone Polyketide

描述（由申请人提供）：模块化聚酮酶（PKS）是合成许多结构复杂和医学上重要的天然产物的显著的多功能酶组件。在过去的十年中，6-脱氧腺苷酸B合酶（DEBS）已成为最好理解的模块PKS。在一定程度上，由于该赠款的持续支持，现在可以以无与伦比的力量询问DEBS的蛋白质化学，酶学和生物合成特性。然而，尽管取得了这些进展，我们才刚刚开始揭开DEBS的结构和机制秘密。在接下来的5年时间里，我们将努力从根本上清楚地了解DEBS如何控制亲电和亲核底物特异性以及其六个模块中的每个模块的链阐述。与前两个资助周期的情况一样，我们的研究将充分利用我们在分子生物学、蛋白质化学、酶学、结构生物学和生物合成工具方面的强大和不断增长的基础，并有望产生新的大环内酯类产品，也许最重要的是，鉴于DEBS作为原型模块化PKS的地位，很有可能，这种见解将很快在其他PKS的背景下得到检验，如果得到证实，将用于扩大"非天然"天然产物的库。为今后五年提出了以下具体目标： 1.开发定量酶学分析，以询问DEBS模块1和4，这是DEBS中唯一尚不能进行动力学分析的模块; 2.剖析和工程化单个DEBS模块对非天然亲核底物（扩展单元）的特异性; 3.分析和控制DEBS催化循环中模块间链转移和C-C键形成之间的平衡; 4.确定DEBS中蛋白质-蛋白质相互作用的新原理，并阐明它们在维持结构完整性和/或促进催化中的作用; 5. DEBS及其组分的溶液NMR、X射线晶体学和电子显微镜研究;以及 6.翻译特定目标1 - 4的见解，以生产选定的新大环内酯。

DESCRIPTION (provided by applicant): Modular polyketide synthases (PKSs) are remarkable multifunctional enzyme assemblies that synthesize numerous structurally complex and medicinally important natural products. Over the past decade, the 6- deoxyerythronolide B synthase (DEBS) has established itself as the best-understood modular PKS. In part due to the sustained support of this grant, it is now possible to interrogate the protein chemical, enzymological and biosynthetic properties of DEBS with unrivaled power. Notwithstanding this progress however, we are only just beginning to uncover the structural and mechanistic secrets of DEBS. During the next 5-year period, we will strive to obtain a fundamental and clear understanding of how DEBS controls electrophilic and nucleophilic substrate specificity as well as chain elaboration in each of its six modules. As has been the case in both previous funding cycles, our studies will fully exploit our strong and growing foundation of molecular biological, protein chemical, enzymological, structural biological and biosynthetic tools, and are also expected to yield new macrolide products, Perhaps most significantly, given the status of DEBS as an prototypical modular PKS, it is likely that such insights will be quickly tested in the context of other PKSs and, if validated, used to expand the pool of "unnatural" natural products. The following Specific Aims are proposed for the next 5 years: 1. Developing quantitative enzymological assays to interrogate DEBS modules 1 and 4, the only modules of DEBS for which kinetic assays are not yet available; 2. Dissecting and engineering the specificity of individual DEBS modules toward unnatural nucleophilic substrates (extender units); 3. Analyzing and manipulating the balance between intermodular chain transfer and C-C bond formation in the catalytic cycle of DEB S; 4. Identifying new principles of protein-protein interactions in DEBS, and elucidating their role in maintaining structural integrity and/or facilitating catalysis; 5. Solution NMR, X-ray crystallographic, and electron microscopy studies on DEBS and its components; and 6. Translating insights from Specific Aims 1-4 to produce selected new macrolides.