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

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.

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