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The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of <i>S. ‘boulardii’</i>. Here in this study, the potential role of the gene heme oxygenase-1 (<i>HMX1</i>) in probiotic yeast bloodstream-derived infections was studied by generating marker-free <i>HMX1</i> deletion mutants with CRISPR/Cas9 technology from both commercial and clinical <i>S. ‘boulardii’</i> isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of <i>HMX1</i> in <i>S. ‘boulardii’</i> significantly (<i>p</i> < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of <i>S. ‘boulardii’</i> strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.