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<p><i>Streptococcus thermophilus</i> and <i>Streptococcus zooepidemicus </i><i>ar</i>e industrially important lactic-acid bacteria, used as probiotics and as hyaluronic acid producers. However, the limited availability of regulatory tools has constrained metabolic engineering in these species. In this study, a lightweight genetic toolkit was established to enable controllable gene overexpression and chromosomal gene repression. The toolkit comprises (i) a series of short constitutive promoters (~ 55&#xa0;bp) with gradient strengths; (ii) an IPTG-inducible promoter; and (iii) a single stem-loop sRNA (~ 38 nt) capable of repressing translation by approximately 80% without requiring auxiliary protein factors. Application of the IPTG-inducible promoter to regulate an sRNA library targeting carbohydrate-metabolism genes and regulatory factors enhanced exopolysaccharide (EPS) production in <i>S. thermophilus</i> by 1.21-fold and hyaluronic acid production in <i>S. zooepidemicus</i> by 1.13-fold. This study provides a lightweight and efficient genetic toolkit for engineering streptococci metabolism.</p>

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A lightweight toolkit of synthetic promoters and regulatory sRNAs for gene expression regulation in industrial Streptococcus

  • 期刊论文

  • 发表日期：

    2026年3月27日

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  • Tianai Kong,
  • Yanan Sun,
  • Guobin Yin,
  • Guosen Cheng,
  • Jianghua Li,
  • Guocheng Du,
  • Jian Chen,
  • Yang Wang

摘要

Streptococcus thermophilus and Streptococcus zooepidemicus are industrially important lactic-acid bacteria, used as probiotics and as hyaluronic acid producers. However, the limited availability of regulatory tools has constrained metabolic engineering in these species. In this study, a lightweight genetic toolkit was established to enable controllable gene overexpression and chromosomal gene repression. The toolkit comprises (i) a series of short constitutive promoters (~ 55 bp) with gradient strengths; (ii) an IPTG-inducible promoter; and (iii) a single stem-loop sRNA (~ 38 nt) capable of repressing translation by approximately 80% without requiring auxiliary protein factors. Application of the IPTG-inducible promoter to regulate an sRNA library targeting carbohydrate-metabolism genes and regulatory factors enhanced exopolysaccharide (EPS) production in S. thermophilus by 1.21-fold and hyaluronic acid production in S. zooepidemicus by 1.13-fold. This study provides a lightweight and efficient genetic toolkit for engineering streptococci metabolism.