大肠杆菌产生的遗传毒素结肠杆菌素与结直肠癌的发展有关。这种次生代谢产物由一种多蛋白机制合成，主要由非核糖体肽合成酶/聚酮合成酶酶组成。为了解密PKS-NRPS混合酶在结肠杆菌素生物合成的关键步骤中的作用，我们对ClbK大型酶的结构进行了广泛的结构表征。在这里，我们展示了ClbK完整的反式AT PKS模块的晶体结构，显示了混合酶的结构特异性。此外，我们还报道了全长ClbK混合物的SAXS溶液结构，显示了二聚体结构以及几个催化室。这些结果为通过PKS-NRPS混合酶转移结肠杆菌素前体提供了结构框架，并为重新设计PKS-NRPS混合大酶生成多种应用的代谢产物铺平了道路。版权所有©2023 Elsevier Ltd.。保留所有权利。

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.Copyright © 2023 Elsevier Ltd. All rights reserved.