Diffusive escape of intermediates limits the rate enhancement that nanocontainers or macromolecular scaffolds can provide for artificial biocatalytic cascades. Nonribosomal peptide synthetases (NRPSs) naturally form gigantic assembly lines and prevent escape by covalently tethering intermediates. Here, we have built DNA-templated NRPS (DT-NRPS) by adding zinc-finger tags to split NRPS modules. The zinc fingers direct the NRPS modules to 9-bp binding sites on a DNA strand, where they form a catalytically active enzyme cascade. Geometric constraints of the DT-NRPSs were investigated using the template DNA as a molecular ruler. Up to four DT-NRPS modules were assembled on DNA to synthesize peptides. DT-NRPSs outperform previously reported DNA-templated enzyme cascades in terms of DNA acceleration, which demonstrates that covalent intermediate channeling is possible along the DNA template. Attachment of assembly line enzymes to a DNA scaffold is a promising catalytic strategy for the sequence-controlled biosynthesis of nonribosomal peptides and other polymers.

中间体的扩散逃逸限制了纳米容器或大分子支架可为人工生物催化级联提供的速率增强。非核糖体肽合成酶 (NRPS) 自然形成巨大的装配线，并通过共价连接中间体来防止逃逸。在这里，我们通过添加锌指标签来拆分 NRPS 模块来构建 DNA 模板化 NRPS (DT-NRPS)。锌指将 NRPS 模块引导至 DNA 链上的 9 bp 结合位点，在那里它们形成具有催化活性的酶级联反应。使用模板 DNA 作为分子标尺研究了 DT-NRPS 的几何约束。多达四个 DT-NRPS 模块组装在 DNA 上以合成肽。DT-NRPSs 在 DNA 加速方面优于先前报道的 DNA 模板酶级联反应，这表明共价中间通道沿着 DNA 模板是可能的。将装配线酶连接到 DNA 支架上是非核糖体肽和其他聚合物的序列控制生物合成的有前途的催化策略。