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

UNLABELLED: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-025-01850-x.