Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.

工程生物合成装配线可以彻底改变生物活性天然产品类似物的可持续生产。尽管酵母展示是一种经过验证的强大工具，可用于改变非核糖体肽合成酶 (NRPS) 中看门人腺苷酸化结构域的底物特异性，但尚未描述这些大酶其他成分的类似策略。在这里，我们报告了一种用于设计负责肽延伸的缩合（C）结构域的高通量方法。我们证明，在酵母上以功能形式展示的 120 kDa NRPS 模块可以与溶液中提供的上游模块有效地相互作用，产生束缚在酵母表面的酰胺产物。使用该系统筛选大型 C 结构域文库，我们重新编程了表面活性素合成酶模块以接受脂肪酸供体，从而将这种非规范底物的催化效率提高了 40 倍以上。由于 C 结构域可以在 NRPS 中充当选择性过滤器，因此这种方法应该有助于这些分子装配线的精密工程。