Functional nucleic acids for green biosynthesis

以微生物为基础的生产为生产精细化学品和运输燃料提供了一种强大的绿色替代品，同时还限制了人类接触危险的合成化学品和加工。尽管最近许多基于酵母的高价值产品的制备取得了成功，但将复杂的生物合成途径引入酵母中需要平衡酶表达水平，最大限度地减少途径瓶颈，并防止自毒效应。因此，目前的方法需要为每个单独的目标分子投入大量的时间和资源，严重限制了目前发酵生产的普遍性和范围。我的研究计划将是第一个利用核酸工具来改进化学品和生物燃料的生物合成的项目。这项建议概述了开发新型功能核酸工具的研究计划，这些工具(1)提高复杂酶反应的效率；(2)减少对酵母的毒性影响，允许增加效价；(3)为新的化学多样性和增强的功能性提供分子支架。例如，我们将设计一种新的DNA编码生物传感器，以实现对数百万酶变体的高通量荧光筛选。我们还将改造之前描述的合成核酶来动态控制关键酶和代谢途径，以减少副产品。我们将选择与具有高亲和力的靶分子(适配子)特异性结合的功能核酸，以隔离或包裹有毒副产物和中间体。最后，我们将表征用叠氮化物或炔烃等双正交手柄取代小官能团的可能性，从而促进酶促结合到大分子支架中。这将允许许多不同的“点击伙伴”，包括具有增强的化学性质的不同官能团，或在药物设计中有用的更大的生物分子(例如，siRNA，适配子)被快速和具体地安装。*本提案中描述的计划代表了开发严格的功能核酸研究计划所需的基础工作。在接下来的五年里，我的团队将致力于开发这些工具，并将它们应用于一个概念上的、基于酵母的生物合成平台，该平台既具有多样性，又面向目标。未来，我们将把我们的原理验证实验结果应用于产生重要靶点的生物合成途径，包括抗菌和抗癌天然产品、先进燃料和平台化学品。这一研究计划将通过提供实现功能核酸在能源高效和安全的绿色生物合成战略中的应用所需的概念框架和实用工具，为该领域做出贡献。这一策略是创新的，因为功能核酸在许多研究领域都有应用，但令人惊讶的是，在生物合成和绿色化学方面缺乏应用。**

Microbial-based production provides a powerful green alternative for producing fine chemicals and transport fuels while also limiting human exposure to hazardous synthetic chemicals and processing. Despite the recent success of yeast-based preparation of many high-value products, importing complex biosynthetic pathways with numerous synthetic steps into yeast requires balancing enzyme expression levels, minimizing pathway bottlenecks, and preventing self-toxic effects. Thus, current approaches require significant investment in time and resources for each individual target molecule, severely limiting the current generality and scope of fermentative production.******My research program herein will be the first to harness nucleic acid tools for improving biosynthesis of chemicals and biofuels. This proposal outlines a research program for developing novel functional nucleic acids tools that (1) increase efficiency of complex enzymatic reactions; (2) reduce toxic effects to yeast, permitting increased titer; and (3) furnish pathways with molecular scaffolds for new chemical diversity and enhanced functionality. For example, we will design a new class of DNA-encoded biosensors to allow high-throughput fluorescent screening of millions of enzyme variants. We will also adapt a previously-described synthetic ribozymes to dynamically control key enzymes and metabolic pathways to reduce side-products. We will select functional nucleic acids that specifically bind to target molecules with high affinity (aptamers) to sequester or encapsulate toxic side products and intermediates. Finally, we will characterize the potential of replacing small functional groups with biorthogonal handles such as azide or alkyne, facilitating enzymatic incorporation into large molecular scaffolds. This would allow many different “click partners”, including diverse functional groups with enhanced chemical properties, or larger biomolecules useful in drug-design (e.g., siRNA, aptamers) to be rapidly and specifically installed.******The plans described in this proposal represent the groundwork needed for the development of a rigorous functional nucleic acids research program. Over the next five years, my group will aim to develop the tools and apply them towards a conceptually-new yeast-based biosynthesis platform that is both diversity and target oriented. In the future, we will apply the findings from our proof-of-principle experiments to biosynthetic pathways producing important targets, including antimicrobial and anticancer natural products, advanced fuels, and platform chemicals. This research program will contribute to the field by providing the conceptual framework and practical tools needed to realize the application of functional nucleic acids for energy efficient and safe green biosynthesis strategies. The strategy is innovative because functional nucleic acids have found applications in many fields of research, but are surprisingly lacking in biosynthesis and green chemistry. **