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 天然产品是有效的活性，特权支架，形成了我们的治疗武器库的基础， 人类健康的所有领域。天然产品及其类似物的持续开发将提供 涉及具有改善的活性和药理学性质同时降低脱靶效应的化合物。的 单个生物合成酶的生物工程化是产生这种新的二级生物合成酶的一种方法。 代谢物。然而，生物工程的努力往往受到阻碍，由于缺乏基本的了解， 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靶向结构改变的新型次级代谢物 需要详细了解各个生物合成步骤。 脂肪酰基-AMP连接酶（FAALs）是从初级脂肪酸中提取脂肪酸的关键生物合成结构域， 用于掺入更复杂的天然产物支架的代谢。FAAL域通常与 对于顺式或反式的多结构域聚酮化合物脱氢酶通过结构连接区或对接结构域， 分别我们假设这些连接区和对接结构域对脂肪酸的转移至关重要。 酸链的特定长度的下游聚酮合酶结构域，我们可以调节这一点， 通过保持适当的关键要素进行转移。该提案旨在确定控制环境污染的关键残留物， 在模型系统中激活和转移脂肪酸链，以应用于更复杂的途径。作为 我们试图开发一种能够产生这些含脂肪酸代谢物的强大的异源宿主。 在目标I中，我们将使用我们的模型系统烯烃（Ols）合酶来鉴定和调节守门连接子 区域和对接域，控制脂肪酸整合到次级代谢产物中。定向进化 使用细菌双杂交系统的实验将使我们能够剖析在Ols中发现的关键对接结构域 含有反式酶结构的同系物。在互补系统中，我们将执行定向 进化实验靶向顺式OIs糖苷酶的接头区域，并直接评估代谢产物 通过温度选择筛选生产。目的我将揭示模型Ols中的关键结构元素 合成酶用于未来具有类似生物合成逻辑的更复杂天然产物酶的生物工程。 在目的II中，我们建议开发恶臭假单胞菌用于脂肪酸的异源表达- 含有天然产物。我们的模型Ols合酶的途径以及生物合成基因簇 编码米卡霉素产生的基因将在恶臭假单胞菌中表达。的生产和生物工程， 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FAAL-ACP加载模块的生物工程和 micacocidin途径将由我们来自Aim I的工作指导。