Development of a Tool for High‐Efficiency, Markerless and Iterative Genome Editing in <i>Shouchella clausii</i>

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 optimised using cell wall weakening agents, achieving efficiencies comparable to other recalcitrant bacilli. A comprehensive and reusable genetic tool 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 plasmid, employing a two-step pop-in/pop-out integration strategy, has demonstrated high versatility for genome editing in both S. clausii and Bacillus subtilis as evidenced by its successful use in multiple cases: (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 system holds strong potential for the development of next-generation probiotics and synthetic biology applications.