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

建议书摘要 细菌生产各种具有药用价值的天然产品，如有效的抗生素和 化疗药物，其中许多是FDA批准的。两个良好的生物合成系统，模块化 多酮合成酶(PKS)和非核糖体肽合成酶(NRPS)是 药用天然产品。这些PKS和NRPS系统被组织成多条装配线，其中 结构域模块，其中每个模块通过一个酰基单位(PKS)或氨基酸扩展生物合成中间体 (NRPS)。PKS和NRPS通常由基因簇编码，合成的天然产物可以 仅从基因序列就可以预测到。这使得它们成为工程人员非常有吸引力的目标，以创造新的 或故意改变的化合物。 每个PKS或NRPS模块都包含一个载体结构域(CP)来连接途径中间体。在行动之后 一个模块完成后，必须将不断增长的链传递给下一个模块。在以下情况下确保路径保真度 序列模块位于不同的多肽上，多肽末端的“对接结构域”有助于转移 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 模块。最近发现的一种蓝藻产生三种维生素A酰胺，天然产物与 杀死肺癌细胞的能力。值得注意的是，这三种维生素A酰胺是由一种分支杂交物合成的 PKS/NRPS通路由单基因簇编码。通过以下方式启用分支点的中间传输 对接域的自然复制，允许施主模块VATM将其产品递送到VATN， VatQ，或VATS。 在这里，我将测试相互竞争的假设，即停靠域可以被用作工程工具或 CP-酶的选择性是中间体转移的关键。我将描述维生素A酰胺的支点 通过测量VatM与VATN、VatQ和VATS的亲和力，确定VatM与Vatn、VatQ和Vats的亲和力。因为VATN、VATQ和VATS对接 域是相同的，亲和力上的任何差异都将是由于下游模块的组成，这 可能是工程方面的主要障碍。我将通过以下方式测试此工程策略的可行性 在两条不同的路径上的非规范分支。我希望将相同的扩展底域安装在 多个模块将促进通过自然和人工界面的流量。将此策略应用于现有 药物生物合成途径可以同时产生多个类似物，可以筛选出增加的 药效好，副作用少。

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.