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

基本信息

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
项目类别：
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

项目摘要

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.

摘要摘要天然产品具有有效的活跃，特权的脚手架，构成了我们整个治疗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访问具有改善活性和药理特性的化合物，同时降低脱靶效应。这单个生物合成酶的生物工程是产生这种新次级的一种方法代谢物。但是，由于对对负责自然产物生物合成的离散酶转化。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产物需要详细了解单个生物合成步骤。脂肪酰基 - 阳离子连接酶（FAALS）是关键的生物合成结构域，从原发性吸取脂肪酸代谢融入更复杂的天然产物支架。 FAAL域通常是链接的与多域聚酮合成酶合成在顺式中或通过结构接头区域或对接结构域进行的trans中，分别。我们假设这些接头区域和对接域对于脂肪的转移至关重要特定长度的酸链到下游聚酮化合物合酶结构域，我们可以调节通过维护适当的密钥元素进行转移。该建议旨在确定控制的关键残留物脂肪酸链在模型系统中的激活和转移，以应用于更复杂的途径。作为好吧，我们试图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AIM I中，我们将使用我们的模型系统Olefin（OLS）合酶来识别和调节守门链接器控制脂肪酸整合到二级代谢产物中的区域和对接结构域。定向进化使用细菌两性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包含反式酶结构的同源物。在互补系统中，我们将执行指导针对顺as合酶的接头区域的进化实验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，用于具有相似生物合成逻辑的更复杂的天然产物酶的未来生物工程。在AIM II中，我们建议开发假单胞菌，以用于脂肪酸的异源表达包含天然产品。我们的模型OLS合酶以及生物合成基因簇的途径粉状素生产的编码将在P. p. putida中表达。生产和生物工程 FAAL-ACP结构域整合长度不同的酰基链将在此异源宿主中编码为促进工程工作。 OLS合酶中的FAAL-ACP加载模块的生物工程和微甲虫途径将由我们的工作从Aim I中指导。