Diversity Supplement for Engineering an unnatural redox cofactor (uRedox) system for efficient biosynthesis of medicines

以可扩展和可靠的方式获得天然产物衍生药物的需求尚未得到满足。 天然产物是由生物系统产生的小分子，但通常数量很少。代谢 工程有望将这些药用小分子的生物合成途径从它们的天然 生产者转化为异源宿主如细菌和酵母，其可以大规模、低成本、 工业过程。这项提案的长期目标是解决代谢领域的重大挑战 工程，即异常复杂的代谢在细胞中，通过绝缘几乎任何想要的 药物的生物合成途径来自于细胞的其他代谢途径，从而使前者可以被研究和 有效优化。中心假设是，这一目标可以通过建立一个非自然的 体内氧化还原辅因子系统（uRedox）以提供所需的生物合成途径的能量。此设计的灵感源自 自然：分解代谢和合成代谢，两个对立的代谢系统负责分解和合成。 因为它们各自具有指定的 氧化还原辅因子NAD和NADP。这一假设的科学前提已经得到论证： 当在体内使用烟酰胺单核苷酸（NMN）作为非天然氧化还原辅因子时， 药物的生产反应是活跃的，而所有其他干扰反应在宿主的复合体 代谢网络保持沉默。为了将uRedox的原型开发成真正的通用技术， 提出了以下具体目标：（1）开发一个简便、高通量的筛选平台，用于获得 NMN依赖性酶按需和整体。具体地说，将对大肠杆菌进行工程改造，使得 只有具有活性的依赖于NMN的酶的细胞才能存活。结合计算 我们建立的蛋白质设计管道，这个基于生长的选择平台将允许快速定制 （2）利用URedox生产不同的药物;大肠杆菌和酿酒酵母，两个最 重要的工业型号主机，内置NMN池。其他一些生物体内也能积累NMN 将这些细胞及其NMN生物合成途径移植到E. coli和革兰氏阳性菌S.啤酒。两 工程主机将作为实施uRedox的机箱;（3）应用uRedox以满足 代谢工程：保存醛类。许多药用化合物或其生物合成中间体 是醛类，由于被细胞内大量的氧化还原修饰，不能在细胞内稳定存在。 内切酶作为一个概念验证，uRedox将被用来使这些醛修饰酶失去活性，所有 从而保留了苄基异喹啉生物碱生物合成中的关键醛中间体 （BIA），一个由约2，500种天然产物组成的家族，包括重要的抗菌、止咳和镇痛药物。 所提出的方法是创新的，因为它直接针对生命的普遍代谢基础结构， 因此可以在生物医学和合成生物学中产生极其广泛的影响。

There is an unmet need to obtain natural product-derived medicines in a scalable and reliable manner. Natural products are small molecules produced by biological systems, but often in low quantities. Metabolic engineering promises to move the biosynthetic pathways of these medicinal small molecules from their native producers into heterologous hosts such as bacteria and yeast, which can be cultured in large-scale, low-cost, industrial processes. The long-term goal of this proposal is to address the grand challenge in metabolic engineering, namely the extraordinary complexity of metabolism in the cells, by insulating virtually any desired pharmaceutical biosynthetic pathway from the rest of cell metabolism, so that the former can be studied and optimized effectively. The central hypothesis is that this goal can be achieved by establishing an unnatural redox cofactor system (uRedox) in vivo to power the desired biosynthetic pathways. This design is inspired by Nature: Catabolism and anabolism, two opposing metabolic systems responsible for breaking down and building up cell components, respectively, are insulated from each other because they each have a designated redox cofactor, NAD and NADP, respectively. The scientific premise of this hypothesis has been demonstrated: when using nicotinamide mononucleotide (NMN) as an unnatural redox cofactor in vivo, only the engineered, productive reaction for a pharmaceutical was active, while all other interfering reactions in the host's complex metabolic network remained silent. To develop this prototype of uRedox into a truly universal technology, the following specific aims are proposed: (1) Develop a facile, high-throughput selection platform for obtaining NMN-dependent enzymes on demand and en masse. Specifically, Escherichia coli will be engineered so that only cells harboring active NMN-dependent enzymes can survive. In combination with the computational protein design pipeline that we established, this growth-based selection platform will allow rapid customization of uRedox to produce different pharmaceuticals; (2) Develop E. coli and Saccharomyces cerevisiae, two most important industrial model hosts, with a built-in NMN pool. Some other organisms can accumulate NMN inside the cells, and their NMN biosynthetic pathways will be transplanted into E. coli and S. cerevisiae. The two engineered hosts will serve as chassis for implementing uRedox; (3) Apply uRedox to address a major need in metabolic engineering: preserving aldehydes. Many medicinal compounds or their biosynthetic intermediates are aldehydes, which cannot stably exist in cells because they are modified by the cells' numerous redox enzymes. As a proof-of-concept, uRedox will be used to render these aldehyde-modifying enzymes inactive all at once and thereby preserve a key aldehyde intermediate in the biosynthesis of benzylisoquinoline alkaloids (BIAs), a family of ~2,500 natural products including important antibacterial, antitussive, and analgesic drugs. The proposed approach is innovative because it directly targets life's universal metabolic infrastructure and therefore can have extremely broad impacts in biomedicine and synthetic biology.