Homologous Recombination–Mediated Assembly of Multiple DNA Fragments in Bacillus subtilis via Protoplast Transformation and Inducible RecA Expression

We establish Bacillus subtilis (B. subtilis) as an alternative host for gap-repair cloning (GRC), a methodology that has been predominantly applied in Saccharomyces cerevisiae (S. cerevisiae), which requires recovery of assembled constructs and subsequent re-transformation into Escherichia coli for plasmid amplification. To overcome limitations of the native competence system in B. subtilis, which can lead to loss of DNA termini during uptake, we employed a protoplast transformation approach. Protoplasts were prepared under conditions in which the homologous recombinase RecA is upregulated, and the endogenous recA promoter was further replaced with a xylose-inducible promoter to enable controlled RecA expression. Using this system, we achieved efficient and accurate assembly of multiple DNA fragments with homologous overlaps in a single transformation event. Assembly accuracy was validated by junction PCR and full-length nanopore sequencing. Using this approach, we successfully constructed plasmids of up to 26.5 kb, including assemblies containing fragments of the human MYC locus. These results demonstrate that RecA induction enhances homologous recombination after DNA uptake and that B. subtilis provides a practical and efficient chassis for the construction of large DNA molecules, offering a streamlined alternative to existing GRC methodologies in synthetic genomics. • Bacillus subtilis is established as a new host for Gap Repair Cloning (GRC). • Protoplast transformation enables efficient assembly of 17–26.5 kb constructs. • Inducible RecA expression enhances multi-fragment homologous recombination. • Human MYC locus fragments (26.5 kb) assembled in a single transformation step. • DNA assembled in B. subtilis can be directly recovered without host transfer.