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

• 批准号: 10477963
• 负责人: Michael Raymond Rankin
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/10477963
• 关键词: Acyl Carrier Protein; Affinity; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Bacteria; Binding; Bleomycin; Carrier Proteins; Catalytic Domain; Chemicals; Complex; Cyanobacterium; Dactinomycin; Data; Docking; Doxorubicin; Effectiveness; Engineering; Ensure; Enzymes; Exhibits; FDA approved; Fluorescence Polarization; Gene Cluster; Gene Duplication; Gene Order; Genes; Goals; Hand; 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; Proteins; Reaction; Role; Series; Source; System; Tertiary Protein Structure; Testing; Therapeutic; Transplantation; Work; analog; chemotherapeutic agent; combinatorial; cytotoxic; design; feasibility testing; flexibility; fungus; improved; lung cancer cell; non-Native; novel; peptide synthase; polyketide synthase; polypeptide; side effect; tool

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，如有效的抗生素， 化疗剂，其中许多是FDA批准的。两口井生物合成系统，模块化 聚酮酶（PKS）和非核糖体肽合成酶（NRPS）是蛋白质合成的主要来源。 天然药物产品。这些PKS和NRPS系统被组织成多个装配线， 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）。PKS和NRPS通常由基因簇编码，并合成可被生物降解的天然产物。 仅从基因序列就能预测到。这使得它们成为工程师创造新的 或有目的地改变的化合物。 每个PKS或NRPS模块包含一个载体结构域（CP）以连接途径中间体。作用后 当一个模块的链完成时，增长链必须传递给下一个模块。为了确保路径保真度， 序列模块在不同的多肽上，多肽末端的“对接结构域”促进转移 从上游模块的CP到正确的下游模块的第一催化域的中间体 module.最近发现的一种蓝细菌产生三类vatiamides，这是一种天然产物， 杀死肺癌细胞的能力值得注意的是，这三个vatiamides是由一个分支的杂合物合成的。 PKS/NRPS通路由单个基因簇编码。分支点的中间传输通过以下方式启用 对接结构域的自然复制，允许供体模块VatM将其产物递送到VatN， VatQ或VatS。 在这里，我将测试相互竞争的假设，对接域可以作为工程工具开发， CP-酶选择性对中间体转移至关重要。我将描述瓦替酰胺的分支点 通过测量VatM对VatN、VatQ和VatS的亲和力来检测VatM途径。因为VatN、VatQ和VatS对接 结构域是相同的，亲和力的任何差异将是由于下游模块的组成， 可能是工程学的主要障碍。我将测试这个工程策略的可行性， 两条不同的路径中的非典型分支。我希望安装相同的对接域， 多个模块将促进通过自然和人工界面的流量。将这一战略应用于现有的 药物生物合成途径可以同时产生多种类似物， 功效和更少的副作用。