Novel and engineered strong constitutive promoters from Lactococcus cremoris for enhanced gene expression

Abstract

Abstract Lactococcus cremoris is an emerging microbial chassis for food-grade and therapeutic protein production. However, existing promoter collections available for L. cremoris provide only a limited dynamic range, thereby constraining applications that require high transcriptional output without chemical inducers. Here, we identified and characterized a set of autologous L. cremoris promoters and engineered variants with markedly enhanced activities. Initial screening in Escherichia coli enabled rapid detection of functional elements, followed by systematic evaluation in L. cremoris . Computational modelling and targeted editing of 5′ untranslated regions revealed that mRNA secondary structures interfering with ribosome binding reduced translation efficiency, and eliminating these structures markedly increased expression. Moreover, promoter variants carrying mutations in putative cre sites revealed the contribution of CcpA-mediated repression, which was ultimately circumvented using a Δ ccpA background. The resulting promoters (particularly PR1, d60PG2, and dSDPR1) exhibited very high activity in our experimental system and may outperform some previously reported constitutive promoters, maintained high expression under low-copy contexts, and functioned across both lactic acid bacteria species and E. coli . These findings expand the regulatory repertoire of L. cremoris and establish a flexible framework for stable, inducer-free heterologous expression in food-grade and therapeutic biotechnology, including chromosomally integrated engineered probiotics. Key points • Highly active constitutive L. cremoris promoters were identified and engineered • Removal of inhibitory 5′UTRs and relief of CcpA repression enhanced gene expression • Promoter toolbox for L. cremoris, other LAB and E. coli was expanded