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Molecules
Volume 30
Issue 15
10.3390/molecules30153132
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Review

Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms

by
Ola Alessa
1,
Yoshifumi Aiba
1,
Mahmoud Arbaah
1,
Yuya Hidaka
1,
Shinya Watanabe
1,
Kazuhiko Miyanaga
1,
Dhammika Leshan Wannigama
2 and
Longzhu Cui
1,*
1
Division of Bacteriology, Department of Infection and Immunity, School of Medicine, Jichi Medical University, Shimotsuke 329-0498, Japan
2
Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata 990-2292, Japan
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(15), 3132; https://doi.org/10.3390/molecules30153132 (registering DOI)
Submission received: 23 June 2025 / Revised: 20 July 2025 / Accepted: 23 July 2025 / Published: 25 July 2025
Versions Notes

Abstract

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.
Keywords: bacteriophage engineering; synthetic biology; phage assembly and rebooting; phage recombineering; CRISPR-Cas systems; retron-mediated editing; non-replicative phage; host range expansion; antimicrobial delivery platforms; cell-free TXTL systems bacteriophage engineering; synthetic biology; phage assembly and rebooting; phage recombineering; CRISPR-Cas systems; retron-mediated editing; non-replicative phage; host range expansion; antimicrobial delivery platforms; cell-free TXTL systems

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MDPI and ACS Style

Alessa, O.; Aiba, Y.; Arbaah, M.; Hidaka, Y.; Watanabe, S.; Miyanaga, K.; Wannigama, D.L.; Cui, L. Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms. Molecules 2025, 30, 3132. https://doi.org/10.3390/molecules30153132

AMA Style

Alessa O, Aiba Y, Arbaah M, Hidaka Y, Watanabe S, Miyanaga K, Wannigama DL, Cui L. Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms. Molecules. 2025; 30(15):3132. https://doi.org/10.3390/molecules30153132

Chicago/Turabian Style

Alessa, Ola, Yoshifumi Aiba, Mahmoud Arbaah, Yuya Hidaka, Shinya Watanabe, Kazuhiko Miyanaga, Dhammika Leshan Wannigama, and Longzhu Cui. 2025. "Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms" Molecules 30, no. 15: 3132. https://doi.org/10.3390/molecules30153132

APA Style

Alessa, O., Aiba, Y., Arbaah, M., Hidaka, Y., Watanabe, S., Miyanaga, K., Wannigama, D. L., & Cui, L. (2025). Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms. Molecules, 30(15), 3132. https://doi.org/10.3390/molecules30153132

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