Tailored base editing toolkits for functional genomics and metabolic engineering in the halophile Salinivibrio.

The development of synthetic biology in non-model halophiles has been hindered by the lack of efficient genetic tools, limiting their industrial applications. In this study, we report the development of an efficient base editing platform for the industrially promising halophile Salinivibrio sp. TGB4. Both adenine and cytosine base editors (ABE and CBE) were engineered and validated, enabling single, dual, and triple-site substitutions at specific loci. ABE demonstrated high efficiency and broad PAM compatibility, while CBE enabled accurate C-to-T substitutions. Additionally, a novel fusion protein (PmCDA-TadA8e-nCas9-UGI) facilitated cooperative dual-base editing, allowing for simultaneous A→G and C→T edits, which expands the potential for genome engineering in halophiles. Beyond tool development, we applied this system to elucidate the acetate metabolism in Salinivibrio sp. TGB4, identifying acs1 as a predominant gene for acetate assimilation and poly(3-hydroxybutyrate) biosynthesis under the tested conditions. This work establishes a versatile base editing toolkit for halophilic bacteria and demonstrates its utility in metabolic pathway analysis, thereby enhancing the genetic engineering potential of Salinivibrio sp. TGB4 for the biotechnological conversion of low-cost carbon sources into valuable chemicals.