Radical SAM enzyme engineering to produce improved thiopeptide antibiotics

 描述(申请人提供)：抗生素的开发和工业化生产涉及生物化学和合成化学之间的密切相互作用。生物学 在整个进化过程中，系统被选中，通过具有立体选择性催化活性和无与伦比的原子经济性的途径产生高生物活性的产品。然而，天然产物的药理性质通常很差，人们使用合成化学方法将它们修饰成有用的药物。这些合成步骤通常具有低产率的特点，涉及到有毒试剂的使用，并大大增加了药物开发的总成本。因此，通过在非天然底物类似物上操作来绕过这些合成步骤的酶的进化[并执行新的生化转化]对于将有效的天然产物有效地转化为临床有用的药物是重要的。 这项建议概述了一种策略，以颠覆诺西肽的自然生物合成途径，以产生具有更高活性和溶解性的诺西肽类似物。我们已经确定自由基SAM酶NOSL是实现这一目标的理想靶标。自由基SAM酶在许多重要的生物合成途径中执行具有化学挑战性的反应，如NOSL的自由基断裂-重组反应。自由基SAM酶的定向进化还没有进行，[我们将利用这个机会发现调整其反应性的有效策略]。我们的具体目标是：[(1)通过定向进化扩大nosL的底物谱；(2)利用nosL建立有效的技术来揭示新的RS化学和]；(3)将工程nosL基因导入本地生产者，以发酵区域特异性修饰的nosi肽类似物。然后，这些抗生素可能会通过交叉偶联化学进行进一步修饰，以获得具有药理前景的非天然产品。所有的前体都可以用混杂酶色氨酸合成酶进行制备性生产，用既定的技术合成，或者从商业供应商那里购买。

 DESCRIPTION (provided by applicant): The development and industrial production of antibiotics involves an intimate interplay between biological and synthetic chemistries. Biological systems have been selected throughout evolution to generate highly bioactive products through pathways with stereoselective catalytic activity and unmatched atom economy. However, natural products typically have poor pharmacological properties and synthetic chemistry is employed to modify them into useful drugs. These synthetic steps frequently feature low yields, involve the use of toxic reagents, and contribute substantially to the overall cost of drug development. Thus, the evolution of enzymes that circumvent these synthetic steps by operating on unnatural substrate analogues [and perform new biochemical transformations] is important for the efficient conversion of potent natural products into clinically-useful drugs. This proposal outlines a strategy to subvert the natural biosynthetic pathway of nosiheptide, a thiopeptide antibiotic, to generate nosiheptide analogues with improved activity and solubility. We have identified the radical SAM enzyme NosL, as an ideal target for this objective. Radical SAM enzymes perform chemically challenging reactions, such as the radical fragmentation-recombination reaction of NosL, in many important biosynthetic pathways. Directed evolution of radical SAM enzymes has not been performed and [we will use this opportunity to uncover efficient strategies for tuning their reactivity]. Our specific aims are: [(1) To expand the substrte profile of NosL through directed evolution; (2) Use NosL to establish efficient techniques for uncovering new RS chemistry and]; (3) Introduce engineered nosL genes into the native producer to ferment regiospecifically modified nosiheptides analogues. These antibiotics may then be further modified through cross-coupling chemistry to access pharmacologically-promising non-natural products. All precursors may be produced on preparative-scale with the promiscuous enzyme tryptophan synthase, synthesized with established techniques, or purchased from commercial vendors.