Development of a high-efficiency constitutive expression system in Salinivibrio strain for next-generation extremophile engineering

The halophilic bacterium Salinivibrio sp. TGB10 represents an emerging microbial chassis for industrial biotechnology, yet its genetic toolkit remains underdeveloped due to insufficient characterization of regulatory elements. Here, we identified and characterized a novel endogenous promoter (p1) through transcriptomic analysis and functional validation. The 185-bp promoter region, located upstream of a gene encoding a hypothetical protein containing leucine-zipper lipoprotein domain, was found to contain all essential regulatory elements including a functional −10 element, extended −10 motif, and −35 element. Through progressive truncation and ribosome binding site engineering, we precisely mapped these elements and revealed their compensatory interactions in transcription initiation. Next, we developed a series of synthetic promoters by randomizing the spacer region between −35 and −10 elements, achieving expression levels spanning four orders of magnitude in Salinivibrio and three orders in Escherichia coli. The practical utility of p1 promoter was demonstrated through efficient production of industrially relevant β-glucosidase, with optimized fermentation conditions yielding 76.83 U/mL activity. This study provides a critical genetic toolkit for metabolic engineering and synthetic biology applications in Salinivibrio.