Functional nucleic acids for green biosynthesis

微生物生产为精细化学品和运输燃料的生产提供了一个强大的绿色替代方案，同时也限制了人类接触危险合成化学品和加工。尽管最近成功地基于酵母制备了许多高价值产品，但将具有许多合成步骤的复杂生物合成途径导入酵母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，目前的方法需要对每个单独的靶分子进行大量的时间和资源投资，严重限制了目前发酵生产的一般性和范围。我的研究计划将是第一个利用核酸工具来改善化学品和生物燃料的生物合成。该提案概述了开发新型功能性核酸工具的研究计划，这些工具（1）提高复杂酶促反应的效率;（2）减少对酵母的毒性作用，允许增加滴度;（3）为新的化学多样性和增强的功能性提供分子支架。例如，我们将设计一类新的DNA编码生物传感器，以允许对数百万种酶变体进行高通量荧光筛选。我们还将采用先前描述的合成核酶来动态控制关键酶和代谢途径，以减少副产物。我们将选择以高亲和力特异性结合靶分子（适体）的功能性核酸，以隔离或封装有毒副产物和中间体。最后，我们将表征用双正交手柄（如叠氮化物或炔）取代小官能团的潜力，从而促进酶促掺入大分子支架中。这将允许许多不同的“点击伴侣”，包括具有增强的化学性质的不同官能团，或可用于药物设计的较大生物分子（例如，siRNA，适体）被快速和特异性地安装。本提案中描述的计划代表了发展严格的功能性核酸研究计划所需的基础。在接下来的五年里，我的团队将致力于开发这些工具，并将其应用于一个概念上全新的基于酵母的生物合成平台，该平台既具有多样性又具有目标导向性。未来，我们将把原理验证实验的结果应用于产生重要目标的生物合成途径，包括抗菌和抗癌天然产物、先进燃料和平台化学品。本研究计划将有助于该领域提供的概念框架和实用工具，实现能源效率和安全的绿色生物合成策略的功能核酸的应用。该策略是创新的，因为功能性核酸已在许多研究领域中找到应用，但令人惊讶地缺乏生物合成和绿色化学。

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.