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. 2023 Feb 20:11:1136095.
doi: 10.3389/fbioe.2023.1136095. eCollection 2023.

Biocontainment strategies for in vivo applications of Saccharomyces boulardii

Karl Alex Hedin  1 , Vibeke Kruse  1 , Ruben Vazquez-Uribe  1 , Morten Otto Alexander Sommer  1
Affiliations

Biocontainment strategies for in vivo applications of Saccharomyces boulardii

Karl Alex Hedin et al. Front Bioeng Biotechnol. .

Abstract

The human gastrointestinal tract is a complex and dynamic environment, playing a crucial role in human health. Microorganisms engineered to express a therapeutic activity have emerged as a novel modality to manage numerous diseases. Such advanced microbiome therapeutics (AMTs) must be contained within the treated individual. Hence safe and robust biocontainment strategies are required to prevent the proliferation of microbes outside the treated individual. Here we present the first biocontainment strategy for a probiotic yeast, demonstrating a multi-layered strategy combining an auxotrophic and environmental-sensitive strategy. We knocked out the genes THI6 and BTS1, causing thiamine auxotrophy and increased sensitivity to cold, respectively. The biocontained Saccharomyces boulardii showed restricted growth in the absence of thiamine above 1 ng/ml and exhibited a severe growth defect at temperatures below 20°C. The biocontained strain was well tolerated and viable in mice and demonstrated equal efficiency in peptide production as the ancestral non-biocontained strain. In combination, the data support that thi6∆ and bts1∆ enable biocontainment of S. boulardii, which could be a relevant chassis for future yeast-based AMTs.

Keywords: S boulardii; biocontainment; biosafety; engineered microbes; gut micobiome; probiotic yeast.

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Conflict of interest statement

All authors are inventors on a patent filed by DTU on the Biocontainment strategy.

Figures

FIGURE 1
FIGURE 1
Graphical abstract of the implemented biocontainment strategy. (A) Schematic overview of the selection of the biocontainment strain. (B) Yeast cells with disruption of the genes synthesising uracil (URA3), thiamine (THI6), and geranylgeranyl diphosphate synthase (BTS1) can grow in the internal environment containing the essential nutrients and optimal temperature. However, proliferation in external environments lacking the essential nutrients and lower temperatures will be limited.
FIGURE 2
FIGURE 2
Selection of auxotrophic mutation in S. boulardii. (A) Schematic overview of the construction of the auxotrophic strains. To generate the uracil, histidine, and tryptophan auxotroph, a null mutation was introduced in the respective genes, while the genes were deleted to generate the vitamin auxotrophs. Bar plot of mean OD600 after 48 h (B) without the required nutrition supplemented and (C) with the required nutrition supplemented. (D) Bar plot of mean OD600 after 48 h under different concentrations of thiamine. Limit of detection (LOD). Data presented as mean +SEM (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001. One-way ANOVA, Dunnett’s post hoc test with Sb (A) or SbU− (B,C) as reference.
FIGURE 3
FIGURE 3
Constructing temperature-sensitive S. boulardii strains. (A) Graphical illustration of the experimental design to confirm the phenotype. (B) Bar plot of the mean area under the curve (AUC) of 96-h cultivation at 15°C, 20°C and 37°C. (C) Bar plot of the mean doubling time in pH 3, 4, 5 and 6. Limit of detection (LOD). Data presented as mean +SEM (n = 3). Two-way ANOVA, Tukey post hoc test. The different letters (a, b, c, d, e, f, g, and h) above the bars indicate statistically different groups (significance level at p < 0.05).
FIGURE 4
FIGURE 4
Characterisation of the combined cold-sensitive and auxotrophic strain. (A) Stacked bar plot of the percentage of SbU− and SbU− + bts1∆ + thi6∆ in a co-culture experiment with and without thiamine. The co-culture was transferred to a fresh culture every 48 h (n = 5). (B) Stacked bar plot of the % of SbU− and SbU− + bts1∆ + thi6∆ in a co-culture experiment at 15°C, 20°C, and 37°C. The co-culture was transferred to a fresh culture every 24 or 48 h (n = 5). (C) Bar plot of the mean concentration of Exendin-4 (ng/ml) quantified in the supernatant at 24 and 48 h of cultivation (n = 3). Data presented as mean +SEM. * p < 0.05, One-way ANOVA, Dunnett’s post hoc test with SbU− as reference.
FIGURE 5
FIGURE 5
In vivo assessment of the biocontainment strains in antibiotic-treated mice. (A) Graphical scheme of the study design. Male C57BL/6 mice were orally administered with ∼108 cells of S. boulardii daily for five successive days, followed by 6 days of washout. On the 10th day was, the water supplemented with an antibiotic cocktail. The mice were again orally administered with ∼108 cells of S. boulardii daily for five successive days, followed by 34 days of washout. (B) Bar plot of S. boulardii abundance (log10 CFU per gram faeces) in conventional mice with intact microbiome. (C) Step plot of percentage of conventional mice with S. boulardii colonised (LOD ≈ 103 CFU/g). (D) Bar plot of S. boulardii abundance (log10 CFU per gram faeces) in antibiotic-treated mice. (E) Step plot of percentage of antibiotic-treated mice with S. boulardii colonised (LOD ≈50 CFU/g). (F) The body weight (grams) during the whole study. (G) Accumulated food intake (gram) throughout the whole study. Data presented as mean ± SEM (n = 4). Data were analysed with One-way ANOVA (B,D) and Two-way ANOVA (F,G), using Dunnett’s post hoc test with SbU− as reference.
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Grants and funding

This work received funding from The Novo Nordisk Foundation under NNF grant number: NNF20CC0035580, NNF Challenge programme CAMiT under Grant agreement: NNF17CO0028232 and the BestTreat project under European Union’s Horizon 2020 research and innovation programme with the Marie Skłodowska-Curie Grant agreement No 813781.

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