# Development of a Tool for High‐Efficiency, Markerless and Iterative Genome Editing in Shouchella clausii

## TL;DR

A new genetic tool enables efficient and precise genome editing in Shouchella clausii, a spore-forming bacterium with potential in biotherapeutics and biotechnology.

## Contribution

Development of a reusable, Golden Gate-compatible shuttle vector and optimized electroporation protocol for markerless genome editing in Shouchella clausii.

## Key findings

- A hyperosmotic electroporation protocol achieved high transformation efficiency in Shouchella clausii.
- The pM4B522 shuttle vector enabled markerless genome editing with success rates exceeding 60% in multiple genetic modifications.
- The system was successfully transferred to Bacillus subtilis, demonstrating broader applicability across Gram-positive bacteria.

## 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.

Shouchella clausii, a spore‐forming Gram‐positive bacterium with a long‐standing use as probiotic, has long been considered genetically intractable. This study introduces a high‐efficiency electroporation protocol and a versatile Golden Gate‐compatible shuttle vector (pM4B522), enabling precise, scarless and iterative genome editing in both S. clausii and Bacillus subtilis, and unlocking new possibilities for applications in biotherapeutics, industrial biotechnology and environmental science.