Genome synthesis, assembly, and rebooting of therapeutically useful high G+C% mycobacteriophages

Bacteriophages show therapeutic promise for treating bacterial pathogens including nontuberculous mycobacteria (NTM). A major impediment is the paucity of therapeutically useful phages and the great variation in the phage infection profiles, especially among Mycobacterium abscessus clinical isolates. These limitations—together with the abundance of mycobacteriophage genes of unknown function—could be addressed by synthetic genetic construction of viruses in which undesirable genes can be eliminated and genetic payloads can be readily added. However, the relatively high G+C% content of mycobacteriophage genomes (64.1%) can be challenging for DNA synthesis using phosphoramidite chemistry, and the genomes are relatively large (40 to 150 kbp) for assembly and rebooting in a bacterial host. Here, we demonstrate efficient de novo synthesis of high G+C% DNA fragments using terminal deoxynucleotidyl transferase chemistry, the reconstruction of complete mycobacteriophage genomes using High-Complexity Golden Gate Assembly, and efficient rebooting via electroporation into Mycobacterium smegmatis . Using this approach, we synthesized the genomes of phages BPs (41.9 kbp, 66.6% G+C%) and Bxb1 (50.5 kbp, 63.6% G+C%), and constructed variants carrying targeted mutations or added payloads. Synthetic construction of mycobacteriophages and their derivatives expands the phage repertoire for therapeutic development and provides versatile tools for advancing mycobacterial genetics and phage-based clinical applications.