Life Sustaining Enzymes From Non-Natural Sequences

来自非天然序列的生命维持酶

• 批准号: 1947720
• 负责人: Michael Hecht
• 金额: $ 101.81万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1947720&HistoricalAwards=false
• 关键词: Life; Sustaining; Enzymes; Non; Natural; Life; Sustaining; Enzymes; Non; Natural

The creation of novel enzymes that are catalytically active in vitro and biologically functional in vivo is a central goal of synthetic biology. The research covered by this project achieves this goal by designing and characterizing life sustaining enzymes from non-natural amino acid sequences. Achieving this goal is important for three reasons: First, the results of this research enhance understanding of the fundamental molecular underpinnings of life. All life on Earth descended from common ancestry, and therefore all living systems share fundamental similarities. This research examines life-sustaining enzymes that did not descend from common ancestry, but instead are designed, selected, and evolved from non-natural amino acid sequences. Characterization of these novel proteins sheds light on whether enzymes from non-natural sequences simply recapitulate the structures and strategies used by nature, or alternatively (and more excitingly), sustain life using sequences, structures, and mechanisms that differ from those that arose by evolution from common ancestry. Second, achieving the goals of this project will broaden the range of macromolecules that can be used for practical applications. Third, an additional goal of the project focuses on explaining evolution to the broader population via collaborations with humanists to develop a discourse on evolution that is scientifically accurate, while simultaneously appealing to spiritually engaged people.The project will achieve the goal of designing and characterizing life-sustaining enzymes from non-natural amino acid sequences, by pursuing the following steps: (1) The first step is the design of a library of potential active sites into a novel protein. The recent determination of the crystal structure of the first life-sustaining de novo enzyme (Syn-F4) provides a scaffold for the design of a combinatorial collection of side chains that explore millions of alternative catalytic sites in the context of a novel protein structure. (2) This library is then subjected to genetic selections for life sustaining functions that rescue auxotrophic strains of E. coli. (3) Next, a series of genetic assays is employed to elucidate the biological pathway(s) impacted by these novel sequences. The results of these assays point towards metabolic pathways and catalytic steps. (4) Ultimately, enzymatic activity must be confirmed (and quantified) biochemically. Toward this goal, the catalytic properties of the de novo enzymes are elucidated by subjecting purified proteins to kinetic studies with substrates and their analogues. (5) three- dimensional structures of the new proteins are solved, thereby enabling visualization of the active sites of the novel enzymes. These structures guide the construction of mutations to confirm the proposed mechanisms of the novel enzymes. (6) The novel (non-evolved) sequences are subjected to laboratory- based evolution to select enhanced levels of enzymatic activity. Completion of these steps will lead to a collection of novel enzymes that did not arise in nature, but which nonetheless provide activities that sustain lifeThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在体内催化活性的新型酶的创造是合成生物学的核心目标。该项目涵盖的研究通过设计和表征从非天然氨基酸序列中维持酶的生命来实现这一目标。实现这一目标的原因很重要，原因有三个：首先，这项研究的结果增强了对生命基本基础的基本分子基础的理解。地球上的所有生命都来自共同的血统，因此所有生物系统都具有基本的相似性。这项研究检查了没有从共同血统降临的，而是从非天然氨基酸序列设计，选择和进化的，持久的生命维持酶。这些新型蛋白质的表征阐明了来自非天然序列的酶是否只是简单地概括了自然所使用的结构和策略，或者（更令人兴奋），使用序列，结构和机制维持与与共同祖先的逐步演变不同的序列，结构和机制。 其次，实现该项目的目标将扩大可用于实际应用的大分子的范围。第三，该项目的另一个目标重点是通过与人文主义者的合作来解释对更广泛的人群的进化，以开发科学准确的进化论，同时吸引了对精神上参与的人的吸引力。该项目将实现设计和表征生命维持生命的酶的目标，从而，通过procepter otter otter otter of stite noke of STEPS of STEAS（1），该项目是一个新的步骤（1），这是一项（1）的一个步骤（1）。最近确定了第一个持久生命的从头酶（SYN-F4）的晶体结构，为在新型蛋白质结构的背景下探索了数百万个替代性催化位点的组合侧链的组合集合提供了脚手架。 （2）然后，将该文库进行遗传选择，以挽救大肠杆菌的持续性可持续性菌株。 （3）接下来，采用一系列遗传测定来阐明受这些新序列影响的生物学途径。这些测定的结果指向代谢途径和催化步骤。 （4）最终，必须通过生物化学确认（和定量）酶活性。为了实现这一目标，通过将纯化的蛋白质对底物及其类似物进行动力学研究来阐明从头酶的催化特性。 （5）解决了新蛋白的三维结构，从而实现了新酶的活性位点的可视化。这些结构指导突变的构建以确认新酶的提议机制。 （6）新型（非进化）序列受到实验室的进化，以选择增强的酶活性水平。这些步骤的完成将导致一系列新的酶，这些酶在本质上没有出现，但是它提供了维持Lifethis Award的活动，反映了NSF的法定任务，并被认为是值得通过基金会的知识分子和更广泛影响的评估评估的评估来支持的。