Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

噬菌体是对抗生物危害性细菌的有效疗法，这些细菌会迅速产生多药耐药性。然而，噬菌体治疗的常规给药受到缺乏快速生产、安全的生物工程和噬菌体详细表征的阻碍。因此，我们展示了一个全面的无细胞平台，用于对广泛的噬菌体进行个性化生产、瞬时工程和蛋白质组学表征。使用质谱法，我们验证了假设的和非结构蛋白，还可以监测噬菌体组装过程中的蛋白质表达。值得注意的是，几微升的一锅反应产生了有效剂量的噬菌体对抗肠聚集性大肠杆菌(EAEC)、鼠疫耶尔森菌和肺炎克雷伯菌. 通过共表达合适的宿主因子，我们可以将无细胞生产范围扩展到针对革兰氏阳性菌的噬菌体。我们进一步介绍了一种非基因组噬菌体工程方法，该方法仅增加了一个复制周期的功能。总之，我们希望这种无细胞方法能够促进逆向和正向噬菌体工程以及临床级噬菌体的定制生产。