ABSTRACT 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.
Cappella et al. (Sun,) studied this question.