Screening, Characterization, and Engineering of Universal Promoters for Bifidobacterium breve

Bifidobacterium breve is a key early life gut colonizer with important physiological functions; however, functional genomic studies in this species are hindered by the lack of robust genetic tools. In this study, we employed RNA-seq to identify endogenous constitutive promoters from B. breve CCFM683. Two of these promoters exhibited significantly higher activity than the reference promoter in a single-fluorescence reporter assay. Further analysis using a dual-fluorescence system revealed that one promoter also possesses bidirectional activity. Rational engineering generated four promoter variants with substantially enhanced activity. Subsequent sequence analysis identified conserved regions within the core promoter sequences of B. breve. Finally, cross-species evaluation in heterologous bifidobacteria identified one promoter with the broadest applicability. Collectively, this study provides a suite of broadly applicable promoter tools for genetic functional studies and heterologous gene expression in B. breve and related species.