The natural product (NP) class of syrbactins are potent proteasome inhibitors produced by hybrids of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we describe the stepwise reassembly of an entire NRPS/PKS hybrid to produce a new syrbactin derivative by utilizing the recently described “eXchange Unit between Thiolation domains” (XUTs) approach. Remarkably, XUT-based engineering allowed the direct assembly of PKS and NRPS modules to introduce an α,β-unsaturated Michael system in a macrolactam moiety, which represents the inhibitory warhead of syrbactins. The novel derivative was produced in E. coli, isolated, and examined for its ability to inhibit yeast (yCP), human constitutive (cCP), and immunoproteasome (iCP). The engineered NP maintained the inhibitory activities of the syrbactin class but, due to rational modifications, inhibited iCP most strongly. Moreover, analysis of the crystal structure of yCP in complex with the derivative revealed further design strategies for even more specific iCP inhibition.

中文翻译：

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用于免疫蛋白酶体抑制剂生产的 syrbactin megasynthetases 的生物工程


天然产物 （NP） 类 syrbactins 是由非核糖体肽合成酶 （NRPS） 和聚酮合酶 （PKS） 杂交产生的有效蛋白酶体抑制剂。在这里，我们描述了整个 NRPS/PKS 杂交体的逐步重组，以利用最近描述的“硫醇化结构域之间的交换单元”（XUTs） 方法产生新的 syrbactin 衍生物。值得注意的是，基于 XUT 的工程允许 PKS 和 NRPS 模块的直接组装，以在大内酰胺部分引入 α，β-不饱和 Michael 系统，这代表了 syrbactins 的抑制性弹头。在大肠杆菌中 产生新型衍生物，分离并检查其抑制酵母 （yCP） 、人组成型 （cCP） 和免疫蛋白酶体 （iCP） 的能力。工程化 NP 保持了 syrbactin 类的抑制活性，但由于合理的修饰，对 iCP 的抑制作用最强。此外，对 yCP 与衍生物复合物中晶体结构的分析揭示了更特异性 iCP 抑制的进一步设计策略。