Engineering Dynamic Control of Natural Product Biosynthesis in Bacteria

项目摘要 在细菌中工程化天然产物的生物合成代表了一种有前途的策略，以生产这些高价值的 和药学上重要的化合物。然而，要实现经济上可行的生产是相当困难的 也很有挑战性为了应对这一挑战，在细胞中建立人工逻辑和动态控制功能， 模仿生物系统中的自然规律已经发展起来，并成为一种强有力的方法， 能够进行上级微生物生产。这种方法使细胞具有很强的生物学鲁棒性， 它们通过感知细胞和环境的变化来自主调整它们的代谢状态和活动。 变化然而，与同时和正交上下施加的自然规律相比， 由于基因调控靶点多样，目前的动态调控技术主要依赖于简单的基因回路 在有限的一组基因上执行单一功能，无论是上调还是下调，这仍然是基本的 也没那么复杂为了弥合这些差距，开发新的遗传工具并将其用于新的控制 战略是非常需要的。最近，PI的实验室已经实现了天然产物的生物合成， 确定的药物特性和治疗作用，例如4-羟基香豆素和5-羟基色氨酸 在大肠大肠杆菌通过合成生物学、代谢工程和蛋白质工程的方法， 反义RNA工具用于干预细胞代谢以增强植物聚酮的生物合成，例如 作为黄烷酮柚皮素。在这个MIRA提案中，我们的目标是探索一个新的和一般的战略， 更复杂的动态控制网络，大大提高了细菌中天然产物的生物合成。的 动态控制网络应具有实现自主智能化的能力， 调节和下调功能，以响应细胞和环境的变化，并维持 细胞在整个生长阶段处于最佳生产状态，以实现最大的生产效率。我们 4-羟基香豆素、5-羟基色氨酸的生物合成研究进展 反义RNA工具将为我们理解如何有效地建立和 在实际应用中，在单元中实现动态控制网络和决策策略。 具体而言，将开展四个具有不同研究活动的连贯项目，其中包括： 用于上调基因表达的水杨酸、对香豆酸和色氨酸响应启动子; 2） 扩大反义RNA工具用于基因表达的可控下调; 3）开发和 基于启动子和反义RNA的动态控制网络的表征; 4）应用动态控制网络 提高E.杆菌

Project Summary Engineering natural product biosynthesis in bacteria represents a promising strategy to produce these high-value and pharmaceutically important compounds. However, to achieve economically viable production is quite difficult and challenging. To address this challenge, establishing artificial logic and dynamic control functions in cells by mimicking natural regulations in biological systems has been developed and becomes a powerful approach to enable superior microbial production. This approach render great biological robustness to the cells and allows them to autonomously adjust their metabolic states and activities by sensing the cellular and environmental changes. However, compared with natural regulations that exert simultaneous and orthogonal up- and down- regulation on various gene targets, the current dynamic control techniques mainly relies on simple gene circuits to perform mono-function, either up- or down-regulation, on a limited set of genes, which are still rudimentary and less sophisticated. To bridge these gaps, development of new genetic tools and their use in novel control strategies are highly desired. Recently, the PI’s lab has achieved the biosynthesis of natural products with defined pharmaceutical properties and therapeutic effects such as 4-hydroxycoumarin and 5-hydroxytryptophan in E. coli through synthetic biology, metabolic engineering and protein engineering approaches and developed antisense RNA tools for intervening cellular metabolism to enhance the biosynthesis of plant polyketides, such as flavanone naringenin. In this MIRA proposal, we aim at exploring a new and general strategy for developing more sophisticated dynamic control network to greatly enhance natural product biosynthesis in bacteria. The dynamic control network is expected to have the capability of implementing autonomous and intelligent up- regulation and down-regulation functions in response to cellular and environmental changes and maintain the cells at optimal production states throughout all growth stages to achieve maximal production efficiency. Our recent progress on the biosynthesis of 4-hydroxycoumarin, 5-hydroxytryptophan and the development of antisense RNAs tools will serve as the groundwork for us to understand how to efficiently establish and implement the dynamic control network and decision-making strategies in cells for real-world applications. Specifically, four coherent projects with distinct research activities will be pursued, which include: 1) developing salicylic acid, p-coumaric acid and tryptophan responsive promoters for up-regulation of gene expression; 2) expanding antisense RNA tools for controllable down-regulation of gene expression; 3) developing and characterizing promoter and antisense RNA based dynamic control network; 4) applying dynamic control network to improve biosynthesis of 4-hydroxycoumarin, flavanone naringenin and 5-hydroxytryptophan in E. coli.