Novel phage-encoded miniDNases as an inspiration for enzyme-based biotechnological and antimicrobial tools

miniDNases are small, non-immunogenic enzymes that degrade nucleic acids (DNA). These highly stable enzymes hold potential applications in biotechnology (industrial fermentations, biotech enzymes) and biomedicine (antibiofilm agents). Bacterial viruses have been proven to be valuable research objects for the development of novel biotechnological tools, such as restriction enzymes and the CRISPR-Cas technology. The enormous pool of unique proteins with unknown function encoded by bacteriophages is therefore thought to serve as a unlimited source to meet the continuous demand in biotechnology for innovative enzymes. We here present a novel family of nucleases with unique protein sequence derived from bacteriophages belonging to the Phikmvvirus genus specifically infecting the opportunistic pathogen Pseudomonas aeruginosa. The so-called 'miniDNases' have an exceptional small size (< 9 kDa), degrading DNA in a non-specific manner without destroying RNA. Nevertheless, they can be readily produced and purified in E. coli. Moreover, truncation mutants weighing less than 7.5 kDa have already proved to be active, as well as fusion mutants measuring four times the size of the nuclease, making it possible to construct fusion enzymes with improved activity (e.g. with antimicrobial peptides). The miniDNases have attractive properties, being highly thermostable (up to 90°C), active in a broad pH range (1-10) and showing higher resistance to denaturing and salt conditions compared to the commercial available DNase I. Their dependency on divalent cations allows for a straightforward inactivation with the chelator citrate. Furthermore, the 11 available miniDNases provide a natural pool of variants, which have all been subjected to phage-host co-evolution, for enzyme optimization. Together, this unique combination of properties makes them highly attractive for many applications that require the removal of DNA, not only in laboratories, but also on an industrial scale (e.g. pharmaceutical industry) and in medical settings (e.g. antibiofilm agent).