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

摘要摘要 天然产品是强大的活性、特权的支架，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 具有更好的活性和药理特性，同时减少脱靶效应的化合物。这个 个体生物合成酶的生物工程是产生这种新的次生酶的一种方法 代谢物。然而，由于缺乏对生物工程的基本了解，生物工程的努力经常受到阻碍。 负责天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的次级代谢物 需要对各个生物合成步骤有详细的了解。 脂肪酰基-AMP连接酶(FAALs)是生物合成的关键结构域，从初级脂肪酸中提取脂肪酸 更复杂的天然产物支架的新陈代谢。法尔域通常是链接的 对于通过结构连接区或对接结构域在顺式或反式中的多结构域聚酮合成酶， 分别进行了分析。我们推测这些连接区域和对接结构域对于脂肪的转移是至关重要的。 将特定长度的酸链连接到下游的聚酮合成酶结构域，我们可以对此进行调节 通过维护适当的关键要素进行转移。这项提案旨在确定控制 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。AS 我们寻求开发一种强大的异源宿主，能够产生这些含有脂肪酸的代谢物。 在目标I中，我们将使用我们的模型系统烯烃(OLS)合成酶来识别和调节门控连接子 管理脂肪酸整合为次生代谢物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。在一个互补的系统中，我们将执行定向 针对顺式OLS合成酶连接区的进化实验和直接评估代谢物 通过温度选择筛选进行生产。目的我将揭示OLS模型中的关键结构元素 合成酶，用于未来具有类似生物合成逻辑的更复杂的天然产物酶的生物工程。 在AIM II中，我们建议培育恶臭假单胞菌，用于异源表达脂肪酸- 含有天然产物的。我们的模型OLS合成酶以及生物合成基因簇的途径 杀菌素生产的编码将在恶臭假单胞菌中表达。红曲霉菌的生产与生物工程 整合不同长度的酰基链的Faal-ACP结构域将在该异源宿主中编码以 促进工程工作。脂肪脂肪合成酶FAAL-ACP装载模块的生物工程 杀菌素途径将由我们从AIM I开始的工作来指导。

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.