Zhang Hailin, Zhu Ru, Wang Zhaofei, He Ruoting, Zhang Yuran, Luan Ji, Yan Yaxian, Zhang Youming, Wang Hailong
State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, China.
School of Life Sciences, Jining Medical University, Rizhao, Shandong, China.
mBio. 2025 Jun 11;16(6):e0358224. doi: 10.1128/mbio.03582-24. Epub 2025 May 23.
The use of virulent bacteriophages (phages) against pathogenic bacteria has recently attracted considerable interest. The limitations of naturally isolated phages have promoted the development of genome engineering methods to optimize their functions; however, engineering of virulent phage genomes in bacterial hosts remains challenging. Here, we describe a SMART (plitting, odifying, ssembling, and ebooing) method for multiplex genome engineering of virulent phages by plitting the genome into multiple segments cloned and inserted into single-copy bacterial artificial chromosome vectors in to overcome the toxicity of phage gene products, odifying multiple targeted loci in parallel through recombineering, ssembling split segments into an intact genome, and ebooing the recombined phage in bacterial hosts. Using the SMART method, the 39.9-kb T7 phage genome was split into 10 segments, and 8 genomic loci were deleted in parallel to obtain a chassis phage with 10% genome reduction. The insertion capacity of the chassis phage genome and the expression levels of exogenous genes at different loci were evaluated. Finally, a synthetic T7 phage that efficiently lyses widely different bacteria, such as , , and , was constructed by expressing heterologous lysins from and phages. SMART will facilitate the programming and understanding of the genome structure and function of virulent phages.
Unlike temperate phages, which integrate into host genomes and allow time for genetic manipulation, lytic phages rapidly hijack the bacterial machinery and trigger host lysis within minutes, leaving an extremely narrow editing window. Furthermore, virulent phage genes usually encode toxic products that inhibit the growth of bacterial hosts. We developed a SMART (splitting, modifying, assembling, and rebooting) method for multiplex genome engineering of virulent phages. We deleted 3.9 kb sequences distributed across 8 sites in the 39.9 kb genome of the wild-type T7 phage to construct a chassis phage. Synthetic T7 phages expressing heterologous lysin genes were constructed to efficiently lyse different bacteria, such as , or . The SMART method will facilitate targeted modifications of phage genomes for the creation of custom-designed phages with enhanced therapeutic efficacy, broader host specificity, and programmable behaviors.
使用烈性噬菌体对抗病原菌最近引起了相当大的关注。天然分离噬菌体的局限性推动了基因组工程方法的发展以优化其功能；然而，在细菌宿主中对烈性噬菌体基因组进行工程改造仍然具有挑战性。在此，我们描述了一种用于烈性噬菌体多重基因组工程的SMART（分割、修饰、组装和重启）方法，该方法通过将基因组分割成多个片段进行克隆并插入单拷贝细菌人工染色体载体，以克服噬菌体基因产物的毒性，通过重组工程并行修饰多个靶向位点，将分割的片段组装成完整的基因组，并在细菌宿主中重启重组噬菌体。使用SMART方法，将39.9 kb的T7噬菌体基因组分割成10个片段，并并行删除8个基因组位点以获得基因组减少10%的底盘噬菌体。评估了底盘噬菌体基因组的插入能力以及不同位点外源基因的表达水平。最后，通过表达来自和噬菌体的异源溶素构建了一种能有效裂解广泛不同细菌（如、和）的合成T7噬菌体。SMART将有助于对烈性噬菌体的基因组结构和功能进行编程和理解。
与整合到宿主基因组中并留出遗传操作时间的温和噬菌体不同，裂解性噬菌体在数分钟内迅速劫持细菌机制并触发宿主裂解，留下极其狭窄的编辑窗口。此外，烈性噬菌体基因通常编码抑制细菌宿主生长的有毒产物。我们开发了一种用于烈性噬菌体多重基因组工程的SMART（分割、修饰、组装和重启）方法。我们在野生型T7噬菌体39.9 kb基因组中分布于8个位点的序列中删除了3.9 kb，以构建底盘噬菌体。构建了表达异源溶素基因的合成T7噬菌体以有效裂解不同细菌，如、或。SMART方法将有助于对噬菌体基因组进行靶向修饰，以创建具有增强治疗效果、更广泛宿主特异性和可编程行为的定制噬菌体。
