Streptococcus thermophilus and Streptococcus zooepidemicus hold significant industrial value. S. thermophilus is a Generally Recognized As Safe strain for dairy fermentation and probiotic applications, and S. zooepidemicus is a natural producer of hyaluronic acid for cosmetics and medical applications. However, the limited availability of genetic regulatory elements has constrained metabolic engineering efforts in these species. Existing natural promoters are often long and contain undefined regulatory elements, while the widely-used NICE (Nisin-Controlled Gene Expression) system requires a large regulatory module that imposes metabolic burden.
The research team developed a toolkit for gene expression control in industrial streptococci, comprising three core components: (i) a series of short constitutive promoters (~55 bp) with strong activities; (ii) a compact IPTG-inducible promoter system achieving 11.9- and 7.2-fold induction in S. thermophilus and S. zooepidemicus ; and (iii) a single stem-loop sRNA capable of ~80% translational repression.
Synthetic promoters were designed based on conserved motifs identified through enrichment analysis of lactic acid bacterial promoters. Basing on the Pspcp promoter, which best represents these conserved features, synthetic promoters with higher activity were created, with the strongest matching P32’s strength while being shorter. The IPTG-inducible system was built on the Pspcp scaffold, enabling robust dose-dependent induction, and lactose also effectively induced the system.
For gene repression, the compact sRNAs comprise a Rho-independent terminator and a 30-nt target-binding region. Its small size allows rapid library construction by embedding target sequences via PCR, without requiring DNA assembly for plasmid construction or any auxiliary protein for regulatory activity.
To demonstrate practical utility, the toolkit was applied to two production-relevant scenarios. In S. thermophilus , an sRNA library was rapidly constructed targeting 123 genes related to carbohydrate metabolism or regulatory functions, and new targets were identified that increased exopolysaccharide yield. Using the same approach in S. zooepidemicus , an sRNA library targeting 248 genes revealed a new target that enhanced hyaluronic acid production. These results highlight the toolkit’s practical value for metabolic engineering in streptococcal chassis.
The work entitled “ A lightweight toolkit of synthetic promoters and regulatory sRNAs for gene expression regulation in industrial Streptococcus ” was published on Systems Microbiology and Biomanufacturing (published on Mar. 27, 2026).
Systems Microbiology and Biomanufacturing
Experimental study
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A lightweight toolkit of synthetic promoters and regulatory sRNAs for gene expression regulation in industrial Streptococcus
27-Mar-2026