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: 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
• 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

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 细菌生产具有药物价值作为潜在抗生素和 化学治疗剂，其中许多是FDA批准的。两个井生物合成系统，模块化 聚酮化合物合酶（PK）和非核糖体肽合成酶（NRP）是主要来源 制药天然产品。这些PK和NRPS系统被组织成多种多样的组装线 域模块，每个模块通过酰基单元（PK）或氨基酸延伸生物合成中间 （NRP）。 PK和NRP通常由基因簇编码，并合成可以是的天然产品 仅根据基因序列进行预测。这使他们非常有吸引力的工程目标创建新的目标 或故意更改化合物。 每个PKS或NRPS模块都包含一个载体域（CP），以将路径中间的路径束缚。行动之后 模块的完整，必须将生长链传递到下一个模块。确保路径忠诚时 顺序模块在不同的多肽上，在多肽末端的“停靠域”促进转移 从上游模块的CP到正确下游的第一个催化域的中间体 模块。最近发现的蓝细菌生产三类的vatiamides，天然产物，带有 杀死肺癌细胞的能力。值得注意的是，这三个瓦蒂胺剂是由分支杂种合成的 PKS/NRPS途径由单个基因簇编码。分支点处的中间转移由 对接域的自然重复，允许供体模块Vatm将其产品传递到任何一个vatn， VATQ或大桶。 在这里，我将测试可以探索可以作为工程工具探索的竞争假设或 CP-酶选择性对于中间转移至关重要。我将描述vatiamide的分支 通过测量VATM对VATN，VATQ和VATS的亲和力的途径。因为vatn，vatq和vats对接 域是相同的，亲和力的任何差异都将归功于下游模块的组成，该模块的组成 可能是工程学的主要障碍。我将通过创建该工程策略的可行性 在两个不同途径中的非规范分支。我希望在跨越相同的对接域的安装 多个模块将促进横渡天然和人造界面的通量。将此策略应用于现有 药物生物合成途径可以简单地创建多个可以筛选以增加的类似物 效力和更少的副作用。