Engineered Bacterial Promoters Supercharge Gene Expression in Lactococcus cremoris

A team of researchers in Argentina and Spain has unveiled a new collection of powerful genetic switches derived from Lactococcus cremoris, the friendly bacterium behind many cheeses and an increasingly popular platform for producing food-grade and therapeutic proteins. The study, published open access in Applied Microbiology and Biotechnology, describes both naturally occurring promoters and rationally engineered variants that drive exceptionally high, constant gene expression without the need for chemical inducers. For a field long constrained by a shortage of strong regulatory elements in lactic acid bacteria, the work offers a toolkit that could accelerate everything from industrial enzyme production to the design of chromosomally engineered probiotics.

Promoters are DNA sequences that sit upstream of genes and recruit the cellular machinery responsible for transcription, effectively determining how much of a given protein a cell will make. In model organisms such as Escherichia coli, biologists enjoy a rich catalogue of well-characterized promoters spanning a wide dynamic range. In Lactococcus cremoris, however, the available promoter collections have historically offered only limited output, which has constrained applications requiring high transcriptional levels without inducers. Inducible systems, which depend on adding costly chemicals to trigger expression, are often impractical for food applications or for live biotherapeutics that must function inside the body.

The research team, led by Javier Nicolás Garay-Novillo and José Luis Barra at the Universidad Nacional de Córdoba, together with José Ángel Ruiz-Masó and Gloria del Solar at the Centro de Investigaciones Biológicas Margarita Salas in Madrid, took a two-pronged approach. First, they searched the L. cremoris genome for autologous promoters, meaning regulatory sequences native to the organism itself, which are attractive for food-grade applications because they avoid introducing foreign regulatory DNA. Second, they engineered these sequences to squeeze out more activity, guided by computational modelling of how the resulting messenger RNA molecules fold.

A key trick was speed. Rather than screening every candidate directly in L. cremoris, which is slower and more demanding to manipulate than standard laboratory bacteria, the researchers first tested their promoter candidates in E. coli. This initial screening step allowed rapid detection of functional elements before the team moved to systematic evaluation in the lactic acid bacterium itself. The cross-species functionality proved to be a bonus rather than a compromise: several of the final promoters worked not only in L. cremoris but also in other lactic acid bacteria species and in E. coli, making the toolbox unusually portable.

One of the most technically interesting findings concerns the 5′ untranslated region, the stretch of RNA between the start of transcription and the beginning of the protein-coding sequence. Computational modelling revealed that in some of the native promoters, this region folds into secondary structures that physically interfere with the ribosome binding site, the RNA sequence that the ribosome must recognize to begin translation. Even when a promoter generated abundant mRNA, those messages were poorly translated because their ribosome landing pads were hidden inside hairpins. By using targeted editing to eliminate these inhibitory structures, the researchers markedly increased protein output, demonstrating that translation efficiency, not just transcriptional strength, sets the ceiling on expression.

The team also probed a second layer of control: catabolite repression. In many Gram-positive bacteria, the global regulator CcpA binds to specific DNA motifs known as cre sites, silencing genes when preferred carbon sources are available. Promoter variants carrying mutations in their putative cre sites revealed that CcpA-mediated repression was dampening the activity of some candidates. The researchers ultimately circumvented this constraint altogether by working in a strain lacking the ccpA gene, a ΔccpA background generously provided by Professor Oscar Kuipers of the University of Groningen. Relieving catabolite repression unlocked further gains in expression, illustrating how global regulatory networks can silently throttle even the strongest local promoters.

The standouts of the resulting collection are three promoters designated PR1, d60PG2, and dSDPR1. In the team’s experimental system, these elements exhibited very high activity and may outperform some previously reported constitutive promoters for L. cremoris. Importantly, they maintained high expression even in low-copy contexts, meaning they do not depend on being carried on high-copy-number plasmids to deliver strong output. That property matters greatly for real-world applications: high-copy plasmids impose metabolic burdens on host cells, can be genetically unstable, and are often undesirable in regulated or therapeutic settings. Promoters that perform well when integrated into the chromosome or carried on modest plasmids open the door to stable, inducer-free expression in engineered probiotics designed to persist in the gut.

The implications extend across several domains of biotechnology. Lactococcus cremoris is an emerging microbial chassis for producing food-grade proteins, where regulatory frameworks favor systems built entirely from elements native to safe organisms. It is also being explored as a delivery vehicle for therapeutic molecules, including mucosal vaccines and enzymes. A flexible set of strong constitutive promoters gives synthetic biologists the ability to tune expression levels of pathway enzymes, secretion signals, or therapeutic payloads without chemical inducers, reducing cost and complexity. Because the top performers also function in other lactic acid bacteria, such as Lactiplantibacillus plantarum and Weissella paramesenteroides strains used in the study’s cross-species tests, the toolbox may transfer readily to related industrial and probiotic chassis.

Methodologically, the study offers a template for promoter engineering that others can follow. The workflow combined bioinformatic identification of native regulatory elements, rapid heterologous screening in E. coli, computational prediction of mRNA secondary structures, targeted editing of 5′ untranslated regions, and manipulation of global regulation through cre site mutagenesis and a ΔccpA host. Each step addressed a distinct bottleneck in the journey from DNA sequence to protein, and the additive gains from removing translational inhibitors and relieving catabolite repression show how multiple regulatory layers must be considered when maximizing expression. The work was supported by funding from SECYT-UNC, CONICET, and CSIC programs, and the authors report no competing interests.

For the growing community engineering lactic acid bacteria, the message is clear: the regulatory repertoire of L. cremoris is no longer the limiting factor it once was. With promoters such as PR1, d60PG2, and dSDPR1 in hand, researchers can now build expression systems that are simultaneously strong, stable, inducer-free, and compatible with food-grade and therapeutic standards. As engineered probiotics and microbial protein factories move closer to clinical and commercial reality, the humble genetic switches characterized in this study may prove to be among the most consequential tools in the lactic acid bacteria toolbox, quietly turning up the volume on the next generation of microbial cell factories.

Subject of Research: Identification and engineering of strong constitutive promoters in Lactococcus cremoris for enhanced gene expression

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

Article References: Garay-Novillo, J. N., Ruiz-Masó, J. Á., Guendulain, T. V., del Solar, G., & Barra, J. L. (2026). Novel and engineered strong constitutive promoters from Lactococcus cremoris for enhanced gene expression. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14062-y

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14062-y

Keywords: Lactococcus cremoris, constitutive promoters, promoter engineering, gene expression, lactic acid bacteria, CcpA, catabolite repression, 5′ untranslated region, synthetic biology, metabolic engineering, probiotics, biotechnology