Expanding the Library of (Un)Natural Products through Megasynthase Docking Domain Engineering

通过大合酶对接域工程扩展（非）天然产物库

基本信息

批准号：
10693897
负责人：
Michael Raymond Rankin
金额：
$ 3.98万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10693897
关键词：
Acyl Carrier Protein Affinity Amino Acids Anabolism Antibiotics Antineoplastic Agents Bacteria Binding Bleomycin C-terminal Carrier Proteins Catalytic Domain Complex Cyanobacterium Dactinomycin Data Docking Doxorubicin Effectiveness Engineering Ensure Enzymes Exhibits FDA approved Fluorescence Polarization Gene Cluster Gene Duplication Gene Order Genes Goals Hybrids Interferometry Kinetics Libraries Malignant neoplasm of lung Mass Spectrum Analysis Measures Microbe Modeling Molecular Machines Monitor N-terminal Natural Products Pathway interactions Peptides Pharmaceutical Preparations Pharmacologic Substance Physical condensation Plants Productivity Proteins Reaction Role Series Source System Tertiary Protein Structure Testing Therapeutic Transplantation Work analog chemical synthesis chemotherapeutic agent combinatorial cytotoxic design feasibility testing flexibility fungus gene synthesis improved lung cancer cell manufacture non-Native novel peptide synthase polyketide synthase polypeptide side effect tool

项目摘要

Proposal Summary Bacteria manufacture a diverse range of natural products with pharmaceutical value as potent antibiotics and chemotherapeutic agents, many of which are FDA-approved. Two well biosynthetic systems, the modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), are major sources of pharmaceutical natural products. These PKS and NRPS systems are organized into assembly lines of multi- domain modules, where each module extends a biosynthetic intermediate by an acyl unit (PKS) or amino acid (NRPS). PKS and NRPS are generally encoded by gene clusters and synthesize natural products that can be predicted from the gene sequence alone. This makes them very attractive targets for engineering to create new or purposefully altered compounds. Each PKS or NRPS module contains a carrier domain (CP) to tether the pathway intermediate. After the action of a module is complete, the growing chain must be passed to the next module. To ensure pathway fidelity when sequential modules are on different polypeptides, “docking domains” at the polypeptide termini facilitate transfer of the intermediate from the CP of the upstream module to the first catalytic domain of the correct downstream module. A recently discovered cyanobacterium produces three classes of vatiamides, natural products with the ability to kill lung cancer cells. Remarkably, the three vatiamides are synthesized by a branched hybrid PKS/NRPS pathway encoded by a single gene cluster. Intermediate transfer at the branch point is enabled by the natural duplication of a docking domain, allowing the donor module VatM to deliver its product to either VatN, VatQ, or VatS. Here I will test the competing hypotheses that docking domains can be exploited as engineering tools or that CP-enzyme selectivity is critical to intermediate transfer. I will characterize the branch point of the vatiamide pathway by measuring the affinity of VatM for VatN, VatQ, and VatS. Because the VatN, VatQ, and VatS docking domains are identical, any difference in affinity will be due to the composition of the downstream modules, which may be the main obstacle to be engineering. I will test the feasibility of this engineering strategy by creating noncanonical branches in two different pathways. I expect that installation of the same docking domain across multiple modules will facilitate flux through both natural and artificial interfaces. Applying this strategy to existing drug biosynthetic pathways could simultaneously create multiple analogs that could be screened for increased potency and fewer side effects.

提案摘要细菌产生多种具有药用价值的天然产物，如强效抗生素和化疗药物，其中许多已获得 FDA 批准，两个良好的生物合成系统，模块化。聚酮合酶（PKS）和非核糖体肽合酶（NRPS）是这些 PKS 和 NRPS 系统被组织成多条装配线。结构域模块，其中每个模块通过酰基单元 (PKS) 或氨基酸延伸生物合成中间体 (NRPS) 和 NRPS 通常由基因簇编码并合成可被合成的天然产物。仅从基因序列预测，这使得它们对于工程创造新的目标非常有吸引力。或有目的地复合。每个 PKS 或 NRPS 模块都包含一个载体结构域 (CP)，用于在作用后束缚通路中间体。一个模块完成后，必须将不断增长的链传递到下一个模块，以确保路径保真度。连续模块位于不同的多肽上，多肽末端的“对接域”有利于转移从上游模块的 CP 到正确下游的第一个催化结构域的中间体最近发现的蓝细菌产生三类 vatiamides，天然产物值得注意的是，这三种 vatiamides 是由分支杂合体合成的。由单个基因簇编码的 PKS/NRPS 途径在分支点的中间转移是通过以下方式实现的。对接域的自然复制，允许供体模块 VatM 将其产品传递给 VatN， VatQ 或 VatS。在这里，我将测试对接域可以被用作工程工具或 CP-酶选择性对于中间转移至关重要。我将描述 vatiamide 的分支点。通过测量 VatM 对 VatN、VatQ 和 VatS 的亲和力途径，因为 VatN、VatQ 和 VatS 对接。域是相同的，任何亲和力的差异都是由于下游模块的组成造成的，可能是工程化的主要障碍，我将通过创建来测试该工程策略的可行性。我希望在两个不同的路径中安装相同的对接域。多个模块将促进通过自然和人工界面的通量，将该策略应用于现有的。药物生物合成途径可以同时产生多种类似物，可以筛选这些类似物以增加效力和副作用更少。