Alessa Ola, Aiba Yoshifumi, Arbaah Mahmoud, Hidaka Yuya, Watanabe Shinya, Miyanaga Kazuhiko, Wannigama Dhammika Leshan, Cui Longzhu
Division of Bacteriology, Department of Infection and Immunity, School of Medicine, Jichi Medical University, Shimotsuke 329-0498, Japan.
Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata 990-2292, Japan.
Molecules. 2025 Jul 25;30(15):3132. doi: 10.3390/molecules30153132.
Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.
噬菌体是地球上数量最多的生物实体，长期以来一直作为模型系统和治疗工具。合成生物学和基因工程的最新进展彻底改变了定制噬菌体的能力，使其具有超越其自然能力的增强功能。本综述概述了噬菌体合成和功能工程的现状，涵盖体内和体外策略。我们描述了体内方法，如噬菌体重组系统、CRISPR-Cas辅助编辑和基于细菌反转录子的方法，以及合成组装平台，包括基于酵母的人工染色体、吉布森组装、金门组装和iPac组装。此外，我们探讨了使用无细胞转录翻译（TX-TL）系统进行体外重启，该系统为基于细胞的重启提供了一种灵活的替代方案，但对于大基因组或结构复杂的噬菌体效果较差。特别关注为靶向应用设计定制噬菌体，包括通过受体结合蛋白修饰扩大宿主范围、递送抗菌蛋白或CRISPR有效载荷，以及构建用于安全临床使用的生物封闭、非复制性衣壳系统。通过示例，我们强调了这些技术如何使噬菌体转变为可编程杀菌剂、精密诊断工具和药物递送载体。总之，这些进展为下一代抗菌平台和合成微生物学奠定了坚实基础。
