Engineering Dynamic Control of Natural Product Biosynthesis in Bacteria

项目摘要 在细菌中设计天然产物的生物合成是生产这些高价值产品的一种很有前途的策略 以及药用上重要的化合物。然而，要实现经济上可行的生产是相当困难的 而且很有挑战性。为了应对这一挑战，通过在细胞中建立人工逻辑和动态控制功能 在生物系统中模拟自然规则已经被开发出来，并成为一种强有力的方法来 实现卓越的微生物生产。这种方法为细胞提供了极大的生物健壮性，并允许 它们通过感知细胞和环境来自主调整自己的代谢状态和活动 改变。然而，与同时施加和垂直向上和向下的自然调节相比， 对各种基因靶点的调控，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，这些基因仍然是初级的 而且不那么老练。为了弥补这些差距，新的基因工具的开发及其在新控制中的应用 战略是非常受欢迎的。最近，PI的实验室已经实现了天然产物的生物合成 明确的药物特性和疗效，如4-羟基香豆素和5-羟基色氨酸 在大肠杆菌中通过合成生物学、代谢工程和蛋白质工程的方法并开发了 用于干预细胞代谢以促进植物多酮生物合成的反义RNA工具，如 作为黄烷酮柚皮苷元。在这项米拉提案中，我们的目标是探索一种新的和总体的战略，以发展 更复杂的动态控制网络，极大地提高了天然产物在细菌中的生物合成。这个 动态控制网络应具备实现自主化、智能化的能力。 调节和下调功能，以响应细胞和环境的变化，并保持 细胞在所有生长阶段都处于最佳生产状态，以实现最大的生产效率。我们的 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 在单元中实施动态控制网络和决策策略，以满足实际应用。 具体地说，将执行四个具有不同研究活动的连贯项目，其中包括：1)开发 水杨酸、p-香豆酸和色氨酸反应启动子上调基因表达；2) 扩展反义RNA工具以可控地下调基因表达；3)开发和 基于启动子和反义RNA的动态控制网络的特征；4)应用动态控制网络 提高4-羟基香豆素、黄酮柚皮苷元和5-羟色氨酸在大肠杆菌中的生物合成。

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.