Engineered Biosynthesis of Novel Macrolactone Polyketide

描述（由申请人提供）：模块化聚酮合成酶（pks）是一种显著的多功能酶组合，可合成许多结构复杂且具有重要药用价值的天然产物。在过去的十年中，6-脱氧红素内酯B合成酶（DEBS）已经成为最容易理解的模块化PKS。部分由于这项拨款的持续支持，现在有可能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的结构和机理秘密。在接下来的5年里，我们将努力对DEBS如何控制其六个模块中的亲电和亲核底物特异性以及链细化有一个基本而清晰的认识。正如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强大且不断增长的基础，并期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，这些见解很可能会很快在其他PKS的背景下进行测试，如果得到验证，用来扩大“非天然”天然产品的范围。政府为未来5年提出的具体目标如下：

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.