Directed evolution towards bioengineering of fatty acid-activating natural product pathways

脂肪酸激活天然产物途径的生物工程定向进化

• 批准号: 10607101
• 负责人: Audrey Elizabeth Ynigez-Gutierrez
• 金额: $ 6.91万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10607101
• 关键词: Acids; Alkenes; Anabolism; Architecture; Area; Binding Proteins; Binding Sites; Bioinformatics; Biological Models; Biomedical Engineering; Carrier Proteins; Chimera organism; Chimeric Proteins; Complex; Development; Directed Molecular Evolution; Docking; Elements; Engineering; Enzymes; Fatty Acids; Future; Gatekeeping; Gene Cluster; Gene Expression; Generations; Goals; Health; Homologous Gene; Human; Hybrids; Individual; Knowledge; Length; Libraries; Ligase; Link; Logic; Mass Fragmentography; Mass Spectrum Analysis; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; Natural Products; Nuclear Magnetic Resonance; Pathway interactions; Process; Production; Property; Proteins; Pseudomonas putida; Research Proposals; Ribosomes; Structure; Structure-Activity Relationship; System; Temperature; Tertiary Protein Structure; Therapeutic; Translations; Type I Polyketide Synthase; Work; analog; bioactive natural products; experimental study; improved; in vivo; liquid chromatography mass spectrometry; metabolic engineering; metabolomics; natural product derivative; novel; peptide synthase; pharmacologic; polyketide synthase; prevent; protein protein interaction; scaffold; screening; secondary metabolite; yeast two hybrid system

SUMMARY ABSTRACT Natural products are potently active, privileged scaffolds that form the basis of our therapeutic arsenal across all areas of human health. The continued development of natural products and their analogs will provide access to compounds with improved activity and pharmacological properties while decreasing off-target effects. The bioengineering of individual biosynthetic enzymes is one method of generating such novel secondary metabolites. However, bioengineering efforts are often stymied due to a lack of fundamental understanding of the discrete enzymatic transformations responsible for natural product biosynthesis. Likewise, whole pathway metabolic engineering focused on generating novel secondary metabolites with targeted structural alterations requires detailed knowledge of individual biosynthetic steps. Fatty acyl-AMP ligases (FAALs) are pivotal biosynthetic domains that draw fatty acids from primary metabolism for incorporation into more complex natural product scaffolds. The FAAL domains are often linked with multidomain polyketide synthases either in cis or trans via structural linker regions or docking domains, respectively. We hypothesize that these linker regions and docking domains are crucial to the transfer of fatty acid chains of specific lengths to the downstream polyketide synthase domains and that we can modulate this transfer by maintaining the appropriate key elements. This proposal seeks to identify the key residues that control the activation and transfer of fatty acid chains in a model system for application to more complex pathways. As well, we seek to develop a robust heterologous host capable of producing these fatty acid-containing metabolites. In Aim I, we will use our model system olefin (Ols) synthase to identify and modulate the gate-keeping linker regions and docking domains that govern fatty acid integration into secondary metabolites. Directed evolution experiments using the bacterial two-hybrid system will allow us to dissect the key docking domains found in Ols homologs that contain a trans enzymatic structure. In a complementary system, we will perform directed evolution experiments targeted towards the linker regions of cis Ols synthases and directly assess metabolite production via a temperature selection screening. Aim I will uncover the key structural elements in the model Ols synthase for future bioengineering of more complex natural product enzymes with similar biosynthetic logic. In Aim II, we propose to develop Pseudomonas putida for the heterologous expression of fatty acid- containing natural products. The pathways for our model Ols synthase as well as the biosynthetic gene cluster encoding for micacocidin production will be expressed in P. putida. The production and bioengineering of the FAAL-ACP domains to integrate acyl chains of varying length will be encoded in this heterologous host to facilitate engineering efforts. The bioengineering of the FAAL-ACP loading modules in Ols synthase and the micacocidin pathway will be guided by our work from Aim I.

摘要 摘要 天然产品是有效的、独特的支架，构成了我们跨领域治疗手段的基础 人类健康的所有领域。天然产物及其类似物的持续开发将提供途径 具有改善活性和药理特性同时减少脱靶效应的化合物。这 单个生物合成酶的生物工程是产生这种新型次级酶的方法之一 代谢物。然而，由于缺乏对生物工程的基本了解，生物工程的努力常常受到阻碍。 负责天然产物生物合成的离散酶促转化。同样，整个路径 代谢工程专注于产生具有目标结构改变的新型次级代谢产物 需要对各个生物合成步骤的详细了解。 脂肪酰基-AMP 连接酶 (FAAL) 是从初级脂肪酸中提取脂肪酸的关键生物合成结构域 代谢以纳入更复杂的天然产物支架。 FAAL 域经常链接在一起 通过结构接头区域或对接结构域以顺式或反式具有多结构域聚酮化合物合酶， 分别。我们假设这些连接区和对接域对于脂肪的转移至关重要 下游聚酮合酶结构域具有特定长度的酸链，我们可以对其进行调节 通过维护适当的关键要素进行转移。该提案旨在确定控制的关键残留物 模型系统中脂肪酸链的激活和转移，以应用于更复杂的途径。作为 好吧，我们寻求开发一种能够产生这些含脂肪酸代谢物的强大异源宿主。 在目标 I 中，我们将使用我们的模型系统烯烃 (Ols) 合酶来识别和调节守门连接子 控制脂肪酸整合到次级代谢物中的区域和对接结构域。定向进化 使用细菌双杂交系统的实验将使我们能够剖析 Ols 中发现的关键对接域 含有反式酶结构的同系物。在互补系统中，我们将执行定向 针对顺式 Ols 合酶接头区域的进化实验并直接评估代谢物 通过温度选择筛选进行生产。目标我将揭示模型 Ols 中的关键结构元素 合成酶，用于未来生物工程具有类似生物合成逻辑的更复杂的天然产物酶。 在目标 II 中，我们建议开发恶臭假单胞菌来异源表达脂肪酸- 含有天然产物。我们模型 Ols 合酶的途径以及生物合成基因簇 米可球菌素生产的编码将在恶臭假单胞菌中表达。生产和生物工程 用于整合不同长度酰基链的 FAAL-ACP 结构域将在该异源宿主中编码 促进工程工作。 Ols合酶中FAAL-ACP装载模块的生物工程和 micacocidin 途径将受到我们 Aim I 的工作的指导。