Development of a Toolkit for High-Efficiency, Markerless, and Iterative Genome Editing in Shouchella clausii

Shouchella clausii is a spore-forming, Gram-positive bacterium with intrinsic antibiotic resistance and promising potential in biotherapeutics, industrial biotechnology, and environmental applications. Its genetic intractability—due to a rigid cell wall and lack of natural competence—has limited its development as a microbial chassis. To facilitate its genetic transformation, a hyperosmotic electroporation protocol was optimized using cell wall weakening agents, achieving efficiencies comparable to other recalcitrant bacilli. A comprehensive and reusable genetic toolkit was developed centred on a temperature-sensitive E. coli–S. clausii shuttle vector (pM4B522), specifically engineered for compatibility with Golden Gate assembly. The plasmid backbone includes a spectinomycin resistance marker and an integrated red fluorescent protein reporter for transformants selection. A removable AmilCP chromoprotein cassette streamlines the assembly process by enabling blue/white screening in E. coli . This platform has demonstrated its versatility in genome editing for both S. clausii and Bacillus subtilis , as evidenced by its use in several applications: (i) sequential, markerless deletions of the non-essential catabolic genes xylA and lacA in S. clausii DSM 8716, with a success rate exceeding 60%; (ii) replacement of the lacA coding sequence with a GFP coding sequence, resulting in fluorescence induction in lactose-supplemented medium; (iii) introduction of a single-base substitution generating a premature stop codon in lacA , showcasing scar-free point mutagenesis; and (iv) transfer of the system to B. subtilis 168, highlighting its broader applicability across Gram-positive bacteria. Given the precision and scarless nature of these genetic modifications, this toolkit holds strong potential for the development of next-generation probiotics and synthetic biology applications.