Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains

• Published in: Microbial cell factories 2022-12, Vol.21 (1), p.259-259, Article 259
• Main Authors: Kim, Man Su, Jeong, Da-Eun, Choi, Soo-Keun
• Format: Article
• Language: English
• Subjects: Bacillus; Bacillus (Bacteria); Bacillus - genetics; Bacillus integrative plasmid; Chromosomes; Circuits; CRISPR; CRISPR-Cas Systems; Cytosine; E coli; Enzymes; Gene Editing; Gene expression; Genes, Synthetic; Genetic aspects; Genetic modification; Genetic research; Genetic transformation; Genome editing; Genomes; Homologous recombination; Host range; Integrative and conjugative element; Methods; Microbial genetic engineering; Microorganisms; Mutagenesis; Mutation; Plasmids; Plasmids - genetics; Proteins; Replication initiator protein; Strains (organisms); Synthetic gene circuit; Toxicity; Undomesticated Bacillus
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01989-w

Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems. Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains. The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.