Laboratory of Organic Chemistry, ETH Zürich, 8093 Zurich, Switzerland.
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
J Am Chem Soc. 2022 Sep 28;144(38):17567-17575. doi: 10.1021/jacs.2c07013. Epub 2022 Sep 7.
High-throughput engineering has the potential to revolutionize the customization of biosynthetic assembly lines for the sustainable production of pharmaceutically relevant natural product analogs. Here, we show that the substrate specificity of gatekeeper adenylation domains of nonribosomal peptide synthetases can be switched from an α-amino acid to an α-hydroxy acid in a single round of combinatorial mutagenesis and selection using yeast cell surface display. In addition to shedding light on how such proteins discriminate between amino and hydroxy groups, the remodeled domains function in a pathway context to produce α-hydroxy acid-containing linear peptides and cyclic depsipeptides with high efficiency. Site-specific replacement of backbone amines with oxygens by an engineered synthetase provides the means to probe and tune the activities of diverse peptide metabolites in a simple and predictable fashion.
高通量工程有可能彻底改变生物合成流水线的定制化，以实现具有可持续性的药物相关天然产物类似物的生产。在这里，我们展示了非核糖体肽合成酶的门控腺苷酸化结构域的底物特异性可以在一轮组合诱变和酵母细胞表面展示的选择中从α-氨基酸切换到α-羟基酸。除了阐明这些蛋白质如何区分氨基和羟基之外，经过改造的结构域在通路背景下发挥作用，以高效率产生含有α-羟基酸的线性肽和环状 depsipeptide。通过工程合成酶将骨架胺定点替换为氧原子，为以简单且可预测的方式探查和调节各种肽代谢物的活性提供了手段。
