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<p>This study aimed to construct a probiotic <i>Saccharomyces boulardii</i>-based biosensor that can sense and respond to nitrate, a biomarker of gut inflammation. To this end, a portion of the nitrate assimilation pathway in <i>Ogataea polymorpha</i> required for the designed nitrate-responsive genetic circuit was introduced into <i>S. boulardii</i>, which lacks the corresponding pathway. Specifically, the genes involved in nitrate transport (<i>YNT1</i>) and nitrate-responsive transcriptional activation (<i>YNA1</i> and <i>YNA2</i>) were introduced into <i>S. boulardii</i> using the CRISPR/Cas9-based genome editing system to construct the SKSC499 strain. Next, we investigated whether <i>YNT1</i>, <i>YNI1</i>, and <i>YNR1</i> promoters, which are activated by nitrate-bound YNA1/YNA2 transcription factor in <i>O. polymorpha</i>, induced expression of the reporter gene (G418 resistance marker) in the SKSC499 strain in a nitrate concentration-dependent manner. Among the candidate promoters, the <i>YNR1</i> promoter effectively induced growth of the SKSC499 strain only when nitrate was present in the medium containing the G418 antibiotic.</p>

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Development of a nitrate-responsive whole-cell biosensor based on engineered probiotic Saccharomyces boulardii

  • 期刊论文

  • 发表日期：

    2025年3月12日

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  • Woohyuk Lee,
  • Sang-Hun Do,
  • Jonghyeok Shin,
  • Sun-Ki Kim

摘要

This study aimed to construct a probiotic Saccharomyces boulardii-based biosensor that can sense and respond to nitrate, a biomarker of gut inflammation. To this end, a portion of the nitrate assimilation pathway in Ogataea polymorpha required for the designed nitrate-responsive genetic circuit was introduced into S. boulardii, which lacks the corresponding pathway. Specifically, the genes involved in nitrate transport (YNT1) and nitrate-responsive transcriptional activation (YNA1 and YNA2) were introduced into S. boulardii using the CRISPR/Cas9-based genome editing system to construct the SKSC499 strain. Next, we investigated whether YNT1, YNI1, and YNR1 promoters, which are activated by nitrate-bound YNA1/YNA2 transcription factor in O. polymorpha, induced expression of the reporter gene (G418 resistance marker) in the SKSC499 strain in a nitrate concentration-dependent manner. Among the candidate promoters, the YNR1 promoter effectively induced growth of the SKSC499 strain only when nitrate was present in the medium containing the G418 antibiotic.