FIELD

The invention is in the field of genetic engineering of beneficial microorganisms. The invention relates to a genetically engineered cell and compositions comprising said cell, wherein said cell is engineered to be vulnerable to more than one environmental factor, to enhance the biosafety profile of said cell. In addition, said cell may secrete a bioactive molecule, preferably a polypeptide comprising a polypeptide hormone and/or a cell penetrating peptide. The genetically engineered cell is suitable for use as a medicament or nutritional and/or dietary supplement.

BACKGROUND

In the later years there has been an increasing interest in the interactions that occur between the gut microbiota and human metabolism. The finding that the gut microbiota is capable of directly influencing human metabolism and the finding that host-microbiome interactions are crucial in various diseases has sparked an interest in the use of ingested microbes as agents for delivery of bioactive and therapeutic compounds in the gastrointestinal tract, a concept also known as microbiome therapeutics.

One of the challenges presented by the concept of microbiome therapeutics relates to unwanted colonization in the host as well as in the surrounding environment i.e., biocontainment challenges. The challenge is to make sure that the microbes used for microbiome therapeutics do not permanently colonize and/or proliferate uncontrolled inside the gastrointestinal tract, cause opportunistic infections in the host or subject, such as fungemia (Rannikko et al., Fungemia and Other Fungal Infections Associated with Use of Saccharomyces boulardii Probiotic Supplements, Emerg Infect Dis. 2021 Aug; 27(8): 2090- 2096), or spread into and colonize the surrounding natural environment.

Therefore, alongside the growing interest in microbiome therapeutics, there is a growing need for microbial strains which can be used safely, with a more controllable colonization- and proliferation-capacity in a subject's gut and/or in a natural environment following excretion.

Methods for biocontainment of bacterial organisms are well known, while strategies for containing eukaryotic microorganisms are less well described. EP3181682 describes biocontainment strategies for Lactobacillus sp., Streptococcus sp. and Bacteroides sp., where the strains are engineered to be thymidine-auxotrophic. Stirling et al., provides and discusses further strategies for promoting the safety profile of engineered microorganisms such as auxotrophy, introduction of toxic genes, temperature sensitivity and pH sensitivity (Stirling et al., Controlling the Implementation of Transgenic Microbes: Are We Ready for What Synthetic Biology Has to Offer?, Mol Cell. 2020 May 21 ;78(4):614-623).

The present disclosure provides genetically engineered cells with an improved safety profile and optimized strategies for biocontainment of, especially, yeast cells, as well as methods for the engineering of such cells.

SUMMARY

In a first aspect, the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confer cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, S0D2 and MET18, which upon partial or full inactivation confer a reduced oxidative stress tolerance, is/are partially or fully inactivated in said cell, and wherein the cell comprises at least two of the features a)-c).

In preferred embodiments, the cell belongs to the species Saccharomyces boulardii. In further embodiments, at least two of the genes selected from the group BTS1, THI6 and YAP1 are partially or fully inactivated in said cell. In additional embodiments, the genes BTS1, THI6 and YAP1 are partially or fully inactivated in said cell.

In additional aspects, the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and b) the gene(s) BTS1 and/or RE11 , which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, c) one or more gene(s) selected from the group consisting of YAP1 , CAD1 , SOD1 , SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell.

In additional aspects, the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein i) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and ii) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, S0D2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell.

In embodiments, the genetically engineered cell has a reduced viability and/or reduced ability to grow, in

I. a thiamine deprived environment, such as an environment with a thiamine level below 0.01 μg/ml, and/or

II. an oxygenated environment, such as in blood, and/or

III. an environment with a temperature below 30 °C.

In embodiments, the genetically engineered cell has a reduced viability and/or reduced ability to grow, in

I. a thiamine deprived environment, such as an environment with a thiamine level below 0.01 μg/ml, and/or

II. an oxidative environment, such as in blood, and/or

III. an environment with a temperature below 30 °C.

In embodiments, the genetically engineered cell according to the invention further comprises one or more heterologous nucleic acid sequences encoding one or more polypeptides. In embodiments the polypeptide is a polypeptide hormone, such as GLP-1 , or a variant or analogue thereof, such as the GLP-1 analogue Exendin-4. Preferably, the polypeptide hormone is Exendin-4, GLP-I 1-37, GLP17-37, or GLP17-36, with an amino acid sequence according to SEQ ID NOs: 24, 26, 28 or 30, respectively, or a functional homologue of Exendin-4, GLP-11-37, GLP17-37, or GLP17-36, which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 24, 26, 28 or 30. In embodiments, the cell further comprises one or more heterologous nucleic acids sequences encoding one or more cell penetrating peptides. In other embodiments the polypeptide is a cell penetrating peptide, such as a cell penetrating peptide. In embodiments, the cell penetrating peptide (CPP) is selected from the group consisting of Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 comprising of consisting of an amino acid sequence according to SEQ ID NOs 102, 103, 104, 105, 106, 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108, 109, or 110. Preferably the cell penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110.

In additional embodiments, the heterologous nucleic acid further encodes an amino acid construct comprising at least one signal peptide which is operably linked to said one or more polypeptides, wherein said signal peptide is selected from the group consisting of polypeptides with an amino acid sequence according to SEQ ID NOs: 41 , 43, 45, 47 or 48. Preferably, the expression of said heterologous nucleic acid sequence(s) is under the control of one or more promoter and/or terminator element(s) selected from the group consisting of promoter sequences SEQ ID NOs: 31-36 and terminator sequences SEQ ID NOs: 37-39.

The above embodiments of the genetically engineered cell may also be incorporated into the various aspects of the cell disclosed herein.

The present disclosure also relates to a composition comprising the genetically engineered cell according to the invention. Such composition may further comprise one or more adjuvant(s) and/or excipient(s). In addition, the composition may be provided as a tablet, capsule or suppository.

In addition, the present disclosure also relates to use of the genetically engineered cell or composition according to the invention as a dietary supplement.

Furthermore, the present disclosure also relates to an engineered cell or composition according to the invention for use as a medicament. The present disclosure also relates to a method of treatment of metabolic disorders comprising oral administration of an effective dose of a genetically engineered cell as disclosed herein.

The present disclosure further relates to a method of treatment of obesity comprising oral administration of an effective dose of a genetically engineered cell according to the present disclosure.

The present disclosure further relates to a weight loss product comprising a container, such as a tablet, capsule or suppository or another suitable pharmaceutical composition, comprising a genetically engineered cell according to the present disclosure, and optionally instructions for use.

The present disclosure further relates to a dietary supplement comprising a composition as disclosed herein.

The present disclosure further relates a consumable comprising a genetically engineered cell of the present disclosure.

The present disclosure further relates a genetically engineered cell according to the present disclosure, or a composition comprising same, for use in enhancing the permeabilization of epithelial cells in a mammal.

The present disclosure further relates to a method of pre-treatment of a subject, comprising the administration of an effective amount of a genetically engineered cell or composition as disclosed herein, wherein said administration increases the permeability of the intestinal epithelial in said patient.

Further aspects the present disclosure relates to genetically engineered cell, which expresses one or more heterologous cell penetrating peptides.

Additional aspects the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, and d) the cell comprises at least two of features a)-c), and wherein said cell comprises one or more heterologous nucleic acid sequences encoding GLP1 or an analogue thereof.

Further aspects of the present disclosure relate to a genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, S0D2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, and d) the cell comprises at least two of features a)-c), and wherein said cell comprises one or more heterologous nucleic acid sequences encoding a heterologous cell penetrating peptide.

BRIEF DESCRIPTION OF FIGURES

Figure 1

Bar plot of mean ODeoo after culturing of the indicated strain for 48 hours, in absence (A) or presence (B) of the required nutritional supplement. Data presented as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 . One-way ANOVA, Dunnett’s post hoc test with Sb as reference.

Figure 2

Bar plot of mean ODeoo after culturing of the SblT, SbU’+t7/2A and SblT+tft/GA strains for 48 hours under different concentrations of thiamine. Limit of detection (LOD). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 . One-way ANOVA, Dunnett’s post hoc test with SbLT as reference. Figure 3

Growth characteristics of thiamine auxotrophic strain. (A) The streaked out and spotted serial dilution of SbLT+tft/GA strain on plates with and without thiamine. (B) Grown biomass from the undiluted samples of SblT or SbLT+tft/GA strains were spread out on fresh selection plates to verify potential escapers.

Figure 4

Bar plot of the mean area under the curve (AUC) of the optical density over time of 96-hour cultivation at 15 °C, 20 °C and 37 °C of selected temperature sensitive strains (SbLT+re/7A and SbU'+btsf A). Limit of detection (LOD). Data presented as mean ± SEM. Two-way ANOVA, Tukey post hoc test. The different letters (a, b, c, and d) above the bars indicate statistically different groups (significance level at p < 0.05)

Figure 5

Bar plot of the mean doubling time at pH 3, 4, 5 and 6 of selected temperature sensitive strains (SbLT+re/fA and SbLT+btsfA), wherein a high doubling time represents slower cellular growth, and a low doubling time represents faster cellular growth. Data presented as mean ± SEM. Two-way ANOVA, Tukey post hoc test. The different letters (a, b, c, d, e, f, and g) above the bars indicate statistically different groups (significance level at p < 0.05).

Figure 6

Combination of cold-sensitive and auxotrophic strain. (A) Bar plot of the mean OD600 after 48 hours of cultivation of the strains Sb and SbLT+btsf A+thi6Δ with (+) and without (-) thiamine and/or uracil added to the media. (B) Bar plot of the mean area under the curve of 96-hour cultivation of the strain SbU'+btsf A+thi6Δ at 15 °C, 20 °C and 37 °C. Data presented as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 . One-way ANOVA, Dunnett’s post hoc test with SbU- as reference.

Figure 7

Bar plot of the mean supernatant concentration of the heterologous polypeptide Exendin-4 produced by the indicated strains. Data presented as mean ± SEM. Figure 8

Graphical scheme of the study design. The mice were orally administered ~10 ⁸ cells of S. boulardii daily for five successive days, followed by six days washout. On the 10 ^th day, the mice had an antibiotic cocktail supplemented with drinking water. The mice were again orally administered ~10 ⁸ cells of S. boulardii daily for five successive days, followed by a washout period of 34 days.

Figure 9

Bar plot of the mean doubling time (h' ¹ ) under anaerobic (0 %) and microaerobic (0.1 % and 1 %) at pH 6. Data presented as mean ± SEM. Two-way ANOVA, Tukey post hoc test. The different letters (a, b, and c) above the bars indicate statistically different groups (significance level at p < 0.05)

Figure 10

Effect of UV-exposure (mimicking oxidative stress conditions) on growth of SblT and SblT +yaplA strains.

Figure 11

Growth characterization of the multi-layered biocontainment strain at different thiamine concentration. Bar plot of mean ODeoo after 48 hours of cultivation of SblT (grey) and SblT +thi6Δ+bts1A (black) strains under different concentrations of thiamine. Limit of detection (LOD). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01 *** p < 0.001 . All samples analyzed with dependent sample t-test.

Figure 12

Cell toxicity and viability of Caco-2 cells treated with different cell penetrating peptides (CPPs). (A) Images of Cytotox Red dye stained Caco-2 cells after 1 h treatment with CPPs. (B) Quantification of average red counts (dead cells) of Cytotox Red dye stained Caco-2 cells using Incucyte standard analysis. (C) AlamarBlue assay after 1 h treatment with different CPPs. The % live cell values given have been normalized to the control wells (considered as 100% viable). Values are represented as means ± SD, n = 2 (cytotoxicity assay) and n = 3 (cell viability assay). Statistical analysis: Analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test, p < 0.01 as compared to the control group. Figure 13

Effect of CPPs on barrier integrity of intestinal epithelial cells. (A) Pictorial representation of TEER and FITC-dextran translocation assays. (B) Change in TEER values after 1 h treatment with CPPs. (C) Permeability of FITC-dextran (Papp A-B 1CF-6 cm/s) after 1 h treatment with CPPs. Values are presented as means ± SD, n = 3. Statistical analysis: Analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test, p < 0.01 as compared to the control group (no treatment).

Figure 14

Antimicrobial effects of CPPs on gut commensal microbial strains. (A) Heat map showing the antimicrobial effects of the CPPs RRL helix, Shuffle, Penetramax, and PN159 in various concentrations ranging from 0.01 pM to 100 pM against different commensal gut microbial strains. The final OD600 values after 24-hour treatment with CPPs were normalized to values between 0 and 1 , where 1 being full growth (no CPP treatment) and 0 being no growth (media only). (B-E) showing the minimum inhibitory concentrations (MICs) along with their effective permeation concentrations (from permeability study) for the CPPs Shuffle, Penetramax, RRL helix, and PN159 respectively.

Figure 15

In vivo characterisation of S. boulardii yap1A strains injected into the circular system of adult mice. A. Schematic overview of the study plan. Male BALBc mice were iv injected in the tail with 10 ⁵ cells per gram.

Figure 16

Effect of cell-free spent media from CPP producing yeast strains on permeability of FITC- dextran 4 across Caco-2 monolayers. Permeability is in relative fluorescence units and presented as mean with standard error mean. Analysis: ANOVA followed by multiple comparisons test, p=<0.05(*) or p=<0.01(**) as compared to the control strain (Sb empty).

Figure 17

Pictorial depiction of the intestinal permeability study design in vivo in mice.

Figure 18

FITC-dextran levels in plasma from treatment or control group mice as measure of intestinal permeability. Results are expressed in mean values (ng/mL plasma), and error bars indicate SEM of 5 mice (PBS) and 8 mice (Sb empty, Sb RRL helix, Sb Penetramax, and Sb PN159) per group.

Figure 19

FITC-dextran levels in plasma from treatment or control group mice as measure of intestinal permeability. Results are expressed in mean values (ng/mL plasma), and error bars indicate SEM of 6 mice (PBS) and 12 mice (Sb empty and Sb PN159) per group.

DETAILED DESCRIPTION

Microorganisms, including yeasts, commonly referred to as probiotics, have been used to restore the balance of the gut microbial ecosystem and to control pathogenic infections. Probiotics are in general defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In addition, engineering of these probiotics could potentially enhance their health benefits on the host. Probiotic yeast cells, such as Saccharomyces boulardii are optimal for use as microbial therapeutics, as the yeast cells are able to express proteins that are not suited for bacterial expression and are also able to perform unique post-translational modifications to therapeutic proteins, furthermore, they do not contain any antibiotic resistance genes, have no vulnerability to bacteriophages and lower risk of horizontal gene transfer with other yeasts or bacteria in the human body.

Biocontainment

An essential consideration in the design of genetically modified organisms is the unintended spread and proliferation of the genetically modified organisms outside the desired environment. In order to overcome this issue, different biocontainment strategies, such as auxotrophy, temperature sensitivity or pH sensitivity, have been employed to ensure minimal proliferation of the genetically modified microorganism outside the indented host environment.

In the current context, biocontainment relates to efforts made to confine or prevent spread of biological agents, such as bacteria, fungi, viruses, genetically modified organisms and toxins.

In the present context, the term “biocontainment strategies” relates to strategies that can be employed to reduce the ability of yeast strains to survive in environments where the yeast strains are undesired. These environments include but are not limited to environments inside a host, or subject, outside of the gastrointestinal tract, such as for example the blood stream, or organs, such as a heart or kidney and environments outside of a host or subject.

The strategies employed herein limit the viability of yeast cells in environments where they are undesired by reducing their ability to proliferate due to increased vulnerability to environmental factors such as temperature, e.g., temperatures below 37 °C, and oxidative stress and/or metabolic factors, such as reduced ability or inability to synthesize vitamins (auxotrophy).

In particular, these strategies involve combining two or more strategies to obtain biocontainment of a yeast cell, preferably a yeast cell of the Saccharomyces genus, and more preferably, the cell is Saccharomyces boulardii. This is obtained by inducing a combination of cold sensitivity, reduced oxidative stress tolerance and/or thiamine sensitivity. In that regard, cold sensitivity is obtained by partially or fully inactivating one or more gene(s) selected from the group consisting of BTS1 and REI1, reduced oxidative stress tolerance is obtained by partially or fully inactivating one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, and thiamine sensitivity is obtained by partially or fully inactivating one or more thiamine synthesis genes(s) selected from the group consisting of THI2 and THI6. Accordingly, the present invention relates to a genetically engineered cell which is sensitivity to more than one environmental factor, selected from the groups consisting of temperature level, oxygen levels and vitamin level.

Thus, an aspect of the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein

I. one or more thiamine synthesis gene(s) are partially or fully inactivated in said cell, and/or

II. one or more gene(s), which upon partial or full inactivation confer(s) temperature sensitivity are partially or fully inactivated in said cell, and/or

III. one or more gene(s), which upon partial or full inactivation confer(s) a reduced oxidative stress tolerance, are partially or fully inactivated in said cell, and wherein the genetically engineered cell comprises at least two features of the features l)-lll). In embodiments of the present disclosure, the at least two features are selected from the group consisting of feature I), feature II) and feature III).

In preferred embodiments, the URA3 gene and/or the HIS3 gene of the cell is/are further partially or fully inactivated in the genetically engineered cell.

As is shown in example 4 and example 5, combination of inactivation of specific genes that confer sensitivity towards two or more different environmental factors, in particular an abolished thiamine synthesis combined with cold sensitivity, reduces the viability of a genetically engineered cell of the present invention and reduces the ability of said cell to grow outside the intestines of the intended host.

In embodiments, the present disclosure relates to a genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, are partially or fully inactivated in said cell, wherein the cell comprises at least two of features a)-c).

In embodiments of the present disclosure, the genetically engineered cell has been further modified to include at least two features out of the features a)-c). In the current context, features a)-c) are specific embodiments of the generic features I), II) and III), respectively. In the current context, features i)-ii) are specific embodiments of the generic features II) and III), respectively.

In embodiments, the cell of the present invention has a deficient thiamine synthesis and an increased cold sensitivity.

In further embodiments, the present disclosure relates to a genetically engineered cell belonging to the Saccharomyces genus, wherein the thiamine synthesis gene(s) THI2 and/or THI6 and the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confer cold sensitivity, are partially or fully inactivated in said cell. In further additional embodiments of the present invention, the genes THI2 and BTS1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2 and REI1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and REI1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and BTS1 are partially or fully inactivated in said cell.

In embodiments, the cell of the present invention has a deficient thiamine synthesis and a reduced oxidative stress tolerance.

In further embodiments, the present disclosure relates to a genetically engineered cell belonging to the Saccharomyces genus, wherein the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confer a reduced oxidative stress tolerance, are partially or fully inactivated in said cell.

In further additional embodiments of the present invention, the genes THI2 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2 and SOD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2 and SOD2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2 and MET18 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and SOD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and SOD2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6 and MET18 are partially or fully inactivated.

In embodiments, the cell of the present invention has a reduced oxidative stress tolerance and an increased cold sensitivity. In further embodiments, the present disclosure relates to a genetically engineered cell belonging to the Saccharomyces genus, wherein the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confer cold sensitivity, is/are partially or fully inactivated in said cell, and one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is/are partially or fully inactivated in said cell.

In further additional embodiments of the present invention, the genes BTS1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes BTS1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes BTS1 and SOD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes BTS1 and SOD2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes BTS1 and MET18 are partially or fully inactivated. In further additional embodiments of the present invention, the genes REI1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes REI1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes REI1 and SOD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes REI1 and SOD2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes REI1 and MET18 are partially or fully inactivated.

In embodiments, the cell of the present invention has a defective thiamine synthesis, a reduced oxidative stress tolerance, an increased cold sensitivity.

In further embodiments, the present disclosure thus relates to a genetically engineered cell belonging to the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confer cold sensitivity, are partially or fully inactivated in said cell, and c) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confer a reduced oxidative stress tolerance, are partially or fully inactivated in said cell. In further additional embodiments of the present invention, the genes THI2, BTS1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, BTS1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, REI1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, REI1 and YAP1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, BTS1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, BTS1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, REI1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, REI1 and CAD1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, BTS1 and S0D1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, BTS1 and S0D1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, REI1 and S0D1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, REI1 and S0D1 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, BTS1 and S0D2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, BTS1 and S0D2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, REI1 and S0D2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, REI1 and S0D2 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, BTS1 and MET18 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, BTS1 and MET18 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI2, REI1 and MET18 are partially or fully inactivated. In further additional embodiments of the present invention, the genes THI6, REI1 and MET18 are partially or fully inactivated.

In embodiments of the present disclosure, the genetically engineered cell has a reduced viability and/or ability to grow in one or more environments selected from one of 1 )-3);

1 ) a thiamine deprived environment, such as a thiamine level below 0.01 μg/ml, and/or

2) an oxygenated environment, such as but not limited to oxygenated blood, and/or 3) an environment with a temperature below 30 °C, such as below 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C, 21 °C, 20°C, 15°C or such as below 10°C.

In embodiments of the present disclosure, the genetically engineered cell has a reduced viability and/or ability to grow in one or more environments selected from one of 1 )-3);

1 ) a thiamine deprived environment, such as a thiamine level below 0.01 μg/ml, and/or

2) an oxidative environment, such as but not limited to blood, and/or

3) an environment with a temperature below 30 °C, such as below 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C, 21 °C, 20°C, 15°C or such as below 10°C.

In embodiments, the genetically engineered cell has a reduced viability and/or ability to grow in at least two of the environments selected from 1 )-3).

In additional embodiments, the genetically engineered cell further has a reduced viability and/or ability to grow in an uracil and/or histidine deprived environment.

Temperature sensitive variants

In example 2 and Figure 4 of the present disclosure, genetic engineering by deletion of specific genes were shown to provide strains which were temperature sensitive, specifically cold sensitive strains, in the sense that they showed a reduced growth in environments with temperatures below 37°C.

Accordingly, temperature sensitivity, and in that context cold sensitivity is in the present disclosure to be understood as sensitivity toward temperatures lower than 37°C, which is the approximate temperature which is found in the intestines of humans. In that regard lower than 37°C, is e.g., lower than 35°C, 30°C, 25°C, 20°C, 15°C, or such as lower than 10°C.

Increased sensitivity can be measured as a reduced ability to proliferate, form colonies and/or survive under circumstances where the temperature is lower than 37°C, for example between 15-20°C, 20-25°C, 25-30°C and/or 30-35°C. Examples of strains that are temperature sensitive are provided in example 2, wherein the temperatures sensitivity is obtained by deletion of the genes REI1 and/or BTS1, wherein the strains have a reduced ability to grow at temperatures lower than 37°C compared to strains without deletion of the

REI1 and/or BTS1 genes.

Accordingly, a yeast strain of the present disclosure is considered temperature, e.g., cold sensitive when the strain shows a reduced growth compared to the same strain wherein the gene(s) REI1 and/or BTS1 is/are not partially or fully inactivated. Such a comparison is shown in example 2, Figure 4, wherein the growth of strains, in which the genes REI1 or BTS1 were partially or fully inactivated, showed an impaired growth, and reduced ability to maintain a high biomass over time at temperatures lower than 37°C, compared to a strain wherein said genes are not partially or fully inactivated. In that regard, in embodiments, the growth is considered reduced when the growth of a temperature sensitive strain is at least 1 .5-fold, such 3-fold, 5-fold or such as 10-fold lower when the temperature is below 20°C, such as below 15°C, compared to a strain, wherein the gene(s) REI1 and/or BTS1 is/are not partially or fully inactivated. In that regard, growth may be measured by e.g., optical density, number of colonies pr. area, colony forming units (CFU) pr. area or volume, or similar method known to the skilled person. Accordingly, a strain wherein the gene(s) REI1 and/or BTS1 is/are partially or fully inactivated may be considered cold sensitive.

One exemplary advantage of applying cold sensitivity as a biocontainment strategy is that cold sensitive strains that are used as microbiome therapeutics, food ingredient or dietary supplements will be less able to proliferative outside of a host or subject, thus limiting their ability to colonize the natural environments, the strains may enter when leaving their host or subject. This is particularly advantageous when the cold sensitive strains are also genetically engineered (GMOs) organisms that should not be allowed to spread in the natural environment, outside the intended host organism.

In the present disclosure, it has been found that partially or fully inactivating either one of the genes BTS1 and REI1 provides yeast strains that are cold sensitive, i.e. , the strains exhibit significantly reduced survival and growth rates in temperatures below 37°C.

In embodiments, the yeast having a reduced viability and or ability to grow in an environment with a temperature below 35°C, such as 34°C, 33°C, 32°C, 31 °C, 30°C or such as below 25°C, 20°C, or below 15°C, comprises one or more partially or fully inactivated gene(s) selected from the group consisting of BTS1 and REI1, which upon partial or full inactivation confers cold sensitivity of said cell. In Saccharomyces boulardii the BTS1 gene encodes the Geranylgeranyl diphosphate synthase (GGPPS) (SEQ ID NO: 6), involved in geranylgeranylation of small GTP-binding proteins that mediate vesicular traffic. Inactivation of the BTS1 gene leads to cold sensitivity and inhibits the ability of the cell to grow in cold environments. The BTS1 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 5, or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 5.

In Saccharomyces boulardii the REI1 gene encodes the Cytoplasmic pre-60S factor (SEQ ID NO: 8), which is involved in bud growth in the mitotic signalling network. Inactivation of the REI1 gene leads to cold sensitivity and inhibits the ability of the cell to grow in cold environments. The REI1 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 7, or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 7.

In embodiments, the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in the cell of the present invention.

In embodiments, the genes BTS1 and REI1 are partially or fully inactivated in the cell of the present invention. In embodiments, the gene BTS1 is partially or fully inactivated in the cell of the present invention. In embodiments, the gene REI1 is partially or fully inactivated in the cell of the present invention. In embodiments, the BTS1 gene is inactivated. In embodiments, the REI1 gene is inactivated.

Reduced oxidative stress tolerance variants

The onset of oxidative stress in yeast cells generally induces an early response, where preexisting antioxidant defences provide immediate protection against the initial sub-lethal accumulation of reactive oxygen species. Oxidative stress this may be induced in a number of different ways, such as UV-exposure or oxygenation of the growth media or addition of oxidative agents. In that regard, containment of a cell of the present invention may also be obtained by reducing the tolerance towards oxidative stress, such as the oxidative stress induced by exposure to UV light or similar. Several genes have been proposed to reduce the tolerance of Saccharomyces towards oxidative stress. In the present invention the genes which may, individually or in combination reduce the oxidative stress tolerance of the strains are YAP1, SOD1, SOD2, CAD1 and MET18. Accordingly, a yeast strain of the present disclosure is considered to have a reduced oxidative stress tolerance, when the strain shows a reduced growth compared to a strain wherein the genes YAP1, SOD1, SOD2, CAD1 and/or MET18 is/are not disrupted by genetic engineering. Such a comparison is shown in example 3, Figure 10, wherein the growth of strains wherein the oxidative stress tolerance is reduced by deletion of the YAP1 gene showed significantly impaired growth when exposed to light. In that regard, in embodiments, a reduced growth is when the growth of strain with reduced oxidative stress tolerance is at least 2-fold, such 3-fold, 5-fold or such as 10-fold lower compared to a strain wherein the non-perturbed oxidative stress tolerance i.e., wherein none of the genes YAP1, SOD1, SOD2, CAD1 and/or MET18 are deleted. In that regard, growth may be measured by e.g., optical density, number of colonies pr. area, or similar method known to the skilled person.

In the present disclosure, “oxygenated environment” and “oxidative environment” are used interchangeably.

The ability of an engineered strain to grow in an oxygenated/oxidative environment may e.g., be assayed as described above and in the examples, where the strains are grown in an oxygenated media, or in a media which increases the oxidative stress, such as a media with added oxidizing agent, such as e.g., Fe3+ or H2O2, where the sensitivity can be measured by comparing e.g., the growth of the strains, compared to a control strain, where a strain which is engineered to be sensitive to an oxygenated/oxidative environment would show less growth compared to a cell which is not engineered to said oxygenated/oxidative environment.

In Saccharomyces the YAP1 gene encodes the Basic leucine zipper (bZIP) transcription factor (SEQ ID NO: 10). Deletion of the YAP1 gene leads to reduced oxidative stress tolerance and inability of the cell to grow under conditions with increased oxidative stress, such as exposure to light. The YAP1 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 9.

In Saccharomyces the SOD1 gene encodes the cytosolic copper-zinc superoxide dismutase (SEQ ID NO: 14). Deletion of the SOD1 gene leads to sensitivity to a variety of environmental stresses including oxidative stress and reduced ability of the cell to grow under conditions with increased oxidative stress, such as exposure to light. The SOD1 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 13. In Saccharomyces the SOD2 gene encodes the mitochondrial manganese superoxide dismutase (SEQ ID NO: 16). Deletion of the SOD2 gene leads to sensitivity oxidative stress and oxygen toxicity and reduced ability of the cell to grow under conditions with increased oxidative stress, such as exposure to light and increased oxygen levels. The SOD2 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 15.

In Saccharomyces the CAD1 gene, which is a paralogue to YAP1, encodes the AP-1-like basic leucine zipper (bZIP) transcriptional activator (SEQ ID NO: 12). Deletion of the CAD1 gene leads to sensitivity oxidative stress and reduced ability of the cell to grow under conditions with increased oxidative stress, such as exposure to light. The CAD1 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 11 .

In Saccharomyces the MET18 gene, encodes the DNA repair/transcription protein MET18/MMS19 (SEQ ID NO: 18). Deletion of the MET18 gene leads to sensitivity oxidative stress and reduced ability of the cell to grow under conditions with increased oxidative stress, such as exposure to light. In addition, a MET18 deletion also confers methionine auxotrophy. The MET18 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 17.

In embodiments of the present disclosure, one or more of the genes selected from the group consisting of YAP1, SOD1, SOD2, CAD1 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is/are partially or fully inactivated in said cell. In further embodiments of the present disclosure, one or more of the genes selected from the group consisting of YAP1, SOD1, SOD2, CAD1 and MET18, is/are partially or fully inactivated in said cell.

In additional embodiments of the present disclosure, the gene(s) YAP1 and/or CAD1 is/are partially or fully inactivated in said cell. In additional embodiments of the present disclosure, the gene(s) SOD1 and/or SOD2 is/are partially or fully inactivated in said cell. In additional embodiments of the present disclosure, the gene MET18 is partially or fully inactivated in said cell. In additional embodiments of the present disclosure, the gene MET18 is partially or fully inactivated in said cell. Thiamine synthesis deficiency

Thiamine, also known as thiamine and vitamin B1 , is an essential micronutrient that some organisms are capable of synthesizing on their own, while other organisms such as humans must obtain it through their diet. In the present context, the term "thiamine" is intended to be synonymous with all the names, thiamine and vitamin B1 .

Many microorganisms, including several species belonging to the Saccharomyces genus is capable of biosynthesizing thiamine. However, like humans, these yeasts cannot survive without vitamin B1. Therefore, yeasts that are deficient in thiamine synthesis requires thiamine supplementation in the environment to proliferate as is also disclosed in example 1.

In the present disclosure, it has been found, as shown in example 1 , that partially or fully inactivating either one of the genes THI2 and THI6 provides yeast strains that are deficient in thiamine synthesis, i.e. , the strains are unable to grow in media where thiamine is unavailable or present in only low concentrations, such as below 0.01 μg/mL as is exemplified by example 1 (Figure 2).

Accordingly, a yeast strain of the present disclosure is considered thiamine synthesis deficient and/or thiamine auxotroph when the strain shows a reduced growth compared to a strain wherein the thiamine synthesis is not disrupted by genetic engineering. Such a comparison is shown in example 1 , Figure 1 and 2, wherein the growth of strains wherein the thiamine synthesis is disrupted by deletion of the THI2 or THI6 gene showed significantly impaired growth in absence of thiamine in the growth media. In that regard, in embodiments, a reduced growth is considered significant when the growth of a thiamine synthesis deficient strain is at least 3-fold, such 5-fold, 7-fold or such as 10-fold lower compared to a strain wherein the thiamine synthesis is not disrupted by genetic engineering. In that regard, growth may be measured by e.g., optical density, number of colonies pr. area, or similar method known to the skilled person.

In embodiments, the yeast having a reduced viability and or ability to grow in a thiamine deprived environment containing thiamine at a level below 0.01 μg/ml also comprises one or more partially or fully inactivated thiamine synthesis gene(s) selected from the group consisting of THIS and THI2.

The ability of an engineered strain to grow in a thiamine deprived environment may e.g., be assayed as described above and in the examples, where the strains are grown in a media with reduced thiamine content, where the sensitivity can be measured by comparing e.g., the growth of the strains, compared to a control strain.

In embodiments, the thiamine synthesis of the genetically engineered cell of the present invention is partially or fully disrupted in said cell. In further embodiments, the thiamine synthesis of said cell is disrupted by partially or fully inactivating the THI2 and/or THI6 gene of said cell. In embodiments, one or more genes involved in the thiamine synthesis in the genetically engineered cell of the present invention is partially or fully disrupted in said cell. In further embodiments of the present disclosure, the one or more thiamine synthesis gene(s) is/are selected from the group consisting of THI2 and THI6, is partially or fully inactivated in said cell. In further embodiments, the thiamine synthesis genes THI2 and THI6 are partially or fully inactivated in said cell. In further embodiments, the thiamine synthesis gene THI2 is partially or fully inactivated in said cell. In additional embodiments of the present disclosure, the thiamine synthesis gene THI6 is partially or fully inactivated in said cell.

In Saccharomyces boulardii the THI2 gene encodes the transcriptional activator of thiamine biosynthetic genes (SEQ ID NO: 2). Deletion of the THI2 gene leads to auxotrophy and inability of the cell to grow in absence of thiamine. The THI2 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 1 , or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 1.

In Saccharomyces boulardii the THI6 gene encodes the Thiamine-phosphate diphosphorylase and hydroxyethylthiazole kinase (SEQ ID NO: 4) which is required for thiamine biosynthesis. Deletion of the THI6 gene leads to auxotrophy and inability of the cell to grow in absence of thiamine. The THI6 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 3, or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 3.

Uracil synthesis deficiency

In the present invention, a further biocontainment step, is disruption of the uracil synthesis, which in examples 1-6 was used as the strain background for the remaining gene knockout strains. Disruption of the uracil synthesis provides a genotype which is unable to grow, in absence of uracil supplement to the environment. Accordingly, in embodiments, the present invention relates to a genetically engineered cell of the Saccharomyces genus, wherein the uracil synthesis is partially or fully disrupted in said cell, and wherein further, a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, SOD2 and MET18 wh\ch upon partial or full inactivation confers a reduced oxidative stress tolerance, are partially or fully inactivated in said cell, wherein the cell comprises at least two of features a)-c).

In embodiments, the uracil synthesis the genetically engineered cell of the present invention is partially or fully disrupted in said cell. In further embodiments, the uracil synthesis of said cell is disrupted by partially or fully inactivating the URA3 gene of said cell. In further embodiments, the URA3 gene of said cell is partially or fully inactivated. In Saccharomyces boulardii the URA3 gene encodes Orotidine-5'-phosphate (OMP) decarboxylase (SEQ ID NO: 20), which is involved in 'de novo' pyrimidine nucleobase biosynthetic process and UMP biosynthetic process. Deletion of the URA3 gene leads to auxotrophy and inability of the cell to grow in absence of uracil. The URA3 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 19, or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 19.

Histidine synthesis deficiency

In the present invention, a further biocontainment step, is disruption of the histidine synthesis, which in examples 1 was suggested as the strain background for the remaining gene knockout strains. Disruption of the histidine synthesis provides a genotype which is unable to grow, in absence of histidine supplement to the environment.

Accordingly, in embodiments, the present invention relates to a genetically engineered cell of the Saccharomyces genus, wherein the histidine synthesis is partially or fully disrupted in said cell, and wherein further, a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18 wh\ch upon partial or full inactivation confers a reduced oxidative stress tolerance, are partially or fully inactivated in said cell, wherein the cell comprises at least two of features a)-c).

In embodiments, the histidine synthesis the genetically engineered cell of the present invention is partially or fully disrupted in said cell. In further embodiments, the histidine synthesis of said cell is disrupted by partially or fully inactivating the HIS3 gene of said cell. In further embodiments, the HIS3 gene of said cell is partially or fully inactivated. In Saccharomyces boulardii the HIS3 gene encodes the Imidazoleglycerol-phosphate dehydratase (SEQ ID NO: 22), which is involved in 'de novo' histidine biosynthetic process and catalyses the sixth step in histidine biosynthesis. Deletion of the HIS3 gene leads to auxotrophy and inability of the cell to grow in absence of histidine. The HIS3 gene of said cell is a gene comprising the nucleic acid sequence according to SEQ ID NO: 21 , or a functional homologue thereof, which nucleic acid sequence is at least 80% identical, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 21 .

In embodiments the THI2 gene is inactivated in said cell. In embodiments the THI6 gene is inactivated in said cell. In embodiments the YAP1 gene is inactivated in said cell. In embodiments the CAD1 gene is inactivated in said cell. In embodiments the SOD1 gene is inactivated in said cell. In embodiments the SOD2 gene is inactivated in said cell. In embodiments the MET18 gene is inactivated in said cell. In embodiments, the (JR43 gene is inactivated in said cell. In embodiments, the HIS3 gene is inactivated in said cell. In embodiments, one or more of the THI2, THI6, YAP1, CAD1, SOD1, SOD2, MET18, URA3, HIS3 and/or the HIS3 genes are inactivated in said cell. In embodiments the THI6, BTS1 and YAP1 genes are inactivated in said cell. In embodiments the URA3, THI6, BTS1 and YAP1 genes are inactivated in said cell.

Expressed polypeptides

As shown in example 6 and Figure 7, the biocontainment strategy of the present invention may be combined with expression of one or more polypeptides, which have a beneficial effect on the host, once expressed and secreted. In some of the examples of the present disclosure, the polypeptide is a polypeptide hormone, Exendin-4, but the polypeptide may in principle be any molecule synthetized within the cell of the invention, which upon secretion provides a health benefit to the subject. In that regard, a cell of the present invention may express one or more polypeptides which facilitates biosynthesis of one or more bioactive molecules, such as but not limited to vitamins, polypeptide hormones and signal molecules. In embodiments of the invention, the polypeptides are cell penetrating peptides (CPPs).

Accordingly, in embodiments of the present invention, the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more polypeptide(s).

In additional embodiments, the one or more polypeptide is one or more polypeptide hormone. A hormone is a type of signalling molecules in multicellular organisms that are produced in a tissue or organ and transported to one or more different tissues or organs, where they exert their biological activity and thereby regulate physiology and /or behaviour.

In vertebrates, hormones are involved in the regulation of many physiological and behavioural activities such as digestion, metabolism, respiration, sensory perception, sleep, excretion, lactation, stress induction, growth and development, movement reproduction and mood.

Peptide hormones, also known as protein hormones, are peptides or proteins that act as hormones. Examples of polypeptide hormones include, but are not limited to, angiotensin, calcitonin, incretin, insulin, glucagon, glucagon like peptide 1 , glucagon like peptide 2, oxytocin, somastatin, and vasopressin. Preferably, the one or more polypeptide hormones is a GLP-1 analogue, such as but not limited to Exendin-4, GLP-I 1-37, GLP17-37, or GLP17-36.

Glucagon-Like-Peptide 1 (GLP-1 ) is a polypeptide hormone that is derived proglucagon in tissue specific posttranslational processing of proglucagon. GLP-1 acts through the GLP-1 receptor and amongst several physiological properties it promotes insulin secretion in a glucose-dependent manner, making GLP-1 and its analogues a subject of intense investigation as a potential treatment for diabetes mellitus.

GLP-1 receptor activation in the brain has also been linked with neurogenesis and neuroprotective effects such as reduced necrotic and apoptotic signalling, cell death, and dysfunction. In the diseased brain, GLP-1 receptor agonist treatment in disease models is associated with protection against diseases such as Parkinson's disease, Alzheimer's disease, stroke, traumatic brain injury and multiple sclerosis. In the present context the term "GLP-1 analogue" is intended to mean a peptide that acts as a GLP-1 receptor agonist, but which is not identical to GLP-1 . The amino acid sequence of GLP-1 is shown in SEQ ID NO: 26.

Non-limiting examples of GLP-1 analogues are GLP7-37, GLP7-36, exenatide, liraglutide, lixisenatide, albiglutide, semaglutide, dulaglutide, tirzepatide and Exendin-4.

Preferably, the GLP-1 analogues are selected from the group consisting of GLP7-37, GLP7-36 and Exendin-4, with the amino acid sequences according to SEQ ID NO: 28 (GLP7-37), 30 (GLP7-36) and 24 (Exendin-4), respectively, and functional homologues thereof which amino acid sequences is at least 80 % identical to any one of SEQ ID NOs: 28 (GLP7-37), 30 (GLP ₇ . 36) and 24 (Exendin-4). In preferred embodiments, the polypeptide hormone is Exendin-4 with an amino acid sequence according to SEQ ID NO: 24.

Exendin-4 is a 39 amino acid peptide according to SEQ ID NO: 24, that acts as a GLP-1 receptor agonist. Exendin-4 has been from Gila monster (Heloderma suspectum) saliva and has been shown to have a significantly higher half-time than GLP-1 .

Accordingly, in preferred embodiments of the present invention, the genetically engineered cell of the present invention expresses Exendin-4 or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 24.

In additional embodiments, the cell further comprises one or more heterologous nucleic acids sequences encoding one or more cell penetrating peptides.

Cell penetrating peptides

Cell penetrating peptides (CPPs) are peptides that have the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. This ability has found wide use in medicine, where CPPs have been used as drug delivery agents and cellular labelling, where CPPs have been used for delivering contrasting agents to cellular targets. CPPs have also been utilized for both delivery of nucleic acids and proteins.

Cell penetrating peptides may be expressed in a cell of the invention either alone or in combination with expression of a polypeptide hormone. The functional benefit of a cell expressing a cell penetrating peptide is that it enhances the permeability of the epithelial barrier, to ease the transportation of a compound across the epithelial barrier. Thus, such a compound may be expressed in conjunction with a cell penetrating peptide or it may be a compound, peptide, or otherwise not expressed by the cell.

Thus, a cell of the present invention may express both a polypeptide hormone, such as GLP- 1 or analogues thereof and a cell penetrating peptide.

In embodiments, the genetically engineered cell co-expresses a cell penetrating peptide and GLP-1 or an analogue thereof. In embodiments, the cell penetrating peptide and the GLP-1 or analogue thereof is encoded by individual heterologous nucleic acids. In other embodiments, the cell penetrating peptide and the GLP-1 or analogue thereof is encoded by the same heterologous nucleic acid construct.

In embodiments, the cell penetrating peptide is fused to the GLP-1 or analogue thereof, such as via. a linker.

Linkers, such as amino acid or peptidomimetic sequences, may be inserted between two or more polypeptides so that the two or more entities maintain, at least in part, a distinct function or activity.

Linkers may have one or more properties that may include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. In embodiments, the expressed polypeptides, e.g., GLP-1 or analogues thereof, signal peptide and/or cell penetrating peptides, may be fused together to form an isolated molecule. Such isolated molecule may comprise single or multiple copies of each of the components, and in any order. For example, the GLP-1 or analogue thereof may be fused to the N- and C-terminus of a cell penetrating peptide and/or a signal peptide as disclosed herein.

Accordingly, in embodiments of the present invention, the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides. Over the past decades, several proteins and peptides have been developed as biotherapeutic agents for the treatment of various diseases. Even though oral administration is preferred, most of the therapeutic proteins and peptides are administered parenterally. Oral delivery is challenging for therapeutic proteins and peptides because of low stability caused by the acidic pH, proteolytic enzymes in the gastrointestinal (Gl) tract and limited absorption from the Gl tract into the systemic circulation due to barriers formed by the mucus and epithelial cell layers. The intestinal epithelial lining is a crucial biological barrier, which is regulated by tight junctions, that are made up of proteins including occludins, claudins and zonula occludens (ZO-1 and ZO-2). Tight junctions are responsible for closing intercellular gaps, thereby determining the paracellular permeability and epithelial barrier integrity. To overcome these permeability challenges for improving drug uptake, several strategies have been explored, including the use of medium chain fatty acids, biosurfactants, ingestible capsules and cell-penetrating peptides (CPPs).

CPPs are generally amphipathic and cationic peptides consisting of 5-30 amino acids. CPPs are often categorized into three types, based on 1) Origin (synthetic, protein-derived, and chimeric CPPs); 2) Conformation (linear and cyclic CPPs); and 3) Physical-chemical properties (cationic, hydrophobic, and amphipathic CPPs). CPPs are highly diverse and exhibit different physicochemical and biological properties. CPPs are commonly used to enable cellular intake or translocation of themselves or a CPP-drug/peptide conjugate by promoting permeation across the cellular plasma membrane in the context of drug delivery. However, for the trans-epithelial delivery of polypeptide drugs, paracellular route might be more suitable due to two reasons: 1. Lower proteolytic activity and 2. Paracellular spaces are aqueous filled channels through which these drugs prefer to diffuse. Several studies have reported that some CPPs can also promote permeation of various cellular barriers through paracellular route both in vitro and in vivo. This could be either due to the high local concentration of the CPPs, influencing the dynamics of the tight junction proteins or due to cell-penetrating tendency of CPPs, targeting intracellular proteins which are involved in regulation of opening and closing of tight junctions. Due to these characteristics, CPPs have been investigated as permeation enhancers in the context of drug delivery coupled to different cargoes such as peptides, proteins, nucleic acids, nanoparticles, and drug molecules.

Transactivating transcriptional activator (Tat) was the first CPP discovered from Human Immunodeficiency Virus 1 (HIV- 1 ). Since then, more than 1 ,500 CPPs have been identified or synthesized and most of these are defined in the manually curated CPP database (CPPsite 2.0). Besides their application as intracellular and paracellular permeation enhancers, CPPs have also been studied for their antimicrobial properties against several pathogenic bacteria and viruses. In that context, CPPs which seem to exhibit antimicrobial effects on pathogens, may have an impact on commensal gut microbes as well. In recent years, a significant amount of evidence has emerged indicating that the gut microbiome has an important role in human health, as well as progression of metabolic disorders, cardiovascular diseases, and brain disorders. Therefore, disruption of the gut microbiome may have significant effects on the host’s health. For instance, oral administration antibiotics has been shown to affect the composition of the endogenous microbiota. Similarly, CPPs have been studied in the context of antimicrobial peptides and demonstrated to have antimicrobial effects in various studies. Co-administration of CPPs in vivo is required in large concentrations of a mM range. It is thus safe to assume that these CPPs might also have an impact on the gut microbiome. Therefore, it is important to study in vitro the impact of CPPs on gut commensals before these peptides are applied in a pre- clinical or clinical setting.

Despite broad applicability of CPPs, there are not many studies comparing the ability of CPPs to enhance paracellular permeability of the intestinal barrier or their potential toxic effects on intestinal cell viability. Furthermore, there are not many reports on the antimicrobial effects of the CPPs against gut commensal microbes.

Examples 7, 8 and 9 present a comparison of the most well characterized CPPs (Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159) side-by-side in terms of their effects on the integrity of the epithelial intestinal barrier and toxicity on epithelial cells and further provided is an assessment of the antimicrobial effects of the most effective CPPs based on the permeation studies, against multiple representative species of the gut microbiota. 8 out of the 9 CPPs employed in examples 7-9 were chosen based on their successful employment as vectors for in vitro transepithelial or in vivo delivery of peptide and protein cargoes as summarized by Kristensen and Nielsen 2016 (Kristensen, M., and Nielsen, H. M. (2016b). Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals. Tissue Barriers 4 (2), e1178369). In addition, PN159 was selected due to its ability to permeate Caco-2 monolayers through the paracellular route by effective modulation of tight junctions at low concentrations as reported by Bocsik et al in 2019 (Bocsik, A., et al. (2019). Dual action of the PN159/KLAL/MAP peptide: Increase of drug penetration across caco-2 intestinal barrier model by modulation of tight junctions and plasma membrane permeability. Pharmaceutics 11 (2), E73).

Cell penetrating peptides (CPPs) are promising tools to enhance absorption of different drugs, including oral delivery of therapeutic proteins and peptides. However, CPPs have diverse mechanisms of membrane permeation, which are difficult to predict from their physical properties: sequence, molecular weight, and charge. CPPs have been explored for their application as permeation enhancers to improve absorption of drugs through the epithelium of the Gl tract, therefore, it is highly possible that these CPPs might interact with the gut microbiota in the Gl tract. Some studies have pointed out that some CPPs can exhibit antimicrobial activity against bacteria, including pathogens.

However, studies involving CPPs often focus on intracellular uptake and transcellular transport but have not studied their potential enhancement of paracellular transport, which is more suited for transport of oral polypeptide drugs. Regarding the toxicity of CPPs, most studies focus on toxicity on host cells but not on their effects on gut microbiota. It is therefore important to experimentally evaluate and compare their paracellular permeability, potential toxicity against host’s cells and gut and safety profiles of the CPPs.

In the present application we systematically screened for effective CPPs that promote intestinal permeation through paracellular route and have negligible or no harmful effects against human epithelial cells and gut microbial strains.

In embodiments, the one or more polypeptide expressed by a cell is one or more cell penetrating peptides (CPPs).

In aspects the present disclosure also relates to genetically engineered cell which expresses a heterologous cell penetrating peptide. In embodiments the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, is not inactivated in said cell expressing a heterologous cell penetrating peptides. In embodiments the THI6, BTS1 and YAP1 genes are inactivated in said cell, and said cell expresses a cell penetrating peptide. In embodiments the URA3, THI6, BTS1 and YAP1 genes are inactivated in said cell, and said cell expresses a cell penetrating peptide.

In further embodiments, the genetically engineered cell expresses a CPP to enhance permeability of the epithelial intestinal barrier. In additional embodiments, the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides, wherein expression of said one or more cell penetrating peptides enhances the permeability of the epithelial intestinal barrier. In embodiments, the CPPs are selected from the group comprising Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 and functional homologues thereof having an amino acid sequence that is at least 80% identical to any one of SEQ IDs NO: 102, 103, 104, 105, 106, 107, 108, 109, and 110. Preferably, the CPPs are selected from the group comprising Penetramax, Shuffle, RRL helix and PN159 and functional homologues thereof having an amino acid sequence that is at least 80% identical to any one of SEQ IDs NO: 107, 108, 109, and 110. Accordingly, in embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides selected from the group comprising Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159, or a functional homologue thereof comprising or consisting of an amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108, 109, or 110. Preferably, the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides selected from the group consisting of penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110.

Accordingly, in embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 111 , 112, 113, 114, 115, 116, 117, 118 and 119, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to any one of SEQ ID NOs: 111 , 112, 113, 114, 115, 116, 117, 118 and 119. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 111 , or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 111.

In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 112, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 112.

In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 113, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 113. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 114, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 114. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 115, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 115. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 116, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 116. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 117, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 117. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 118, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 118. In embodiments the cell comprises one or more heterologous nucleic acid sequence(s) encoding one or more cell penetrating peptides wherein said nucleic acid comprises a sequence according to SEQ ID NO: 119, or a variant thereof comprising or consisting of a nucleic acid sequence is at least 80 % identical to SEQ ID NO: 119.

Shuffle is a sequence optimized analogue of Penetratin and has been shown to have an improved drug delivery potential, which could be due to the rearrangement of hydrophobic tryptophan residues as it has been demonstrated that tryptophan residues in an amphipathic CPP sequence positively impact on the internalization into cells. Previously, the sequence of Shuffle has been further optimized to synthesize multiple analogues. Out of these analogues, Penetramax significantly improved the intestinal delivery of insulin compared to Shuffle.

Accordingly, in embodiments, the genetically engineered cell of the present invention expresses Penetramax comprising or consisting of an amino acid sequence according to SEQ ID NO: 107, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 107.

In embodiments, the genetically engineered cell of the present invention comprises one or more heterologous nucleic acid sequence(s) encoding Penetramax, comprising or consisting of an amino acid sequence according to SEQ ID NO: 107, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 107.

In embodiments, the genetically engineered cell of the present invention expresses Shuffle comprising or consisting of an amino acid sequence according to SEQ ID NO: 108, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 108.

In embodiments, the genetically engineered cell of the present invention comprises one or more heterologous nucleic acid sequence(s) encoding Shuffle, comprising or consisting of an amino acid sequence according to SEQ ID NO: 108, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 108.

In embodiments, the genetically engineered cell of the present invention expresses RRL helix comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 109.

In embodiments, the genetically engineered cell of the present invention comprises one or more heterologous nucleic acid sequence(s) encoding RRL helix comprising or consisting of an amino acid sequence according to SEQ ID NO: 109, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 109.

In embodiments, the genetically engineered cell of the present invention expresses PN159 comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 110. In embodiments, the genetically engineered cell of the present invention comprises one or more heterologous nucleic acid sequence(s) encoding PN159 comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or a functional homologue thereof having an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 99% or 99.9% identical to SEQ ID NO: 110.

A signal peptide

When secretion of a polypeptide is preferred, the polypeptide, such as a polypeptide hormone, may be operably linked with one or more signal peptides (i.e. , a fusion polypeptide). Signal peptide(s) can function to prompt the host cell to translocate the polypeptide after expression. In general, a signal peptide may direct an expressed polypeptide to a specific cellular compartment and/or organelle or signal for cellular excretion. In the present context, a signal peptide preferably directs the expressed polypeptide to cellular excretion. A signal peptide is typically 16-100 amino acids long and present at the N-terminus of a newly synthesized polypeptide. In relation to the nucleic acid construct of the invention, the signal peptide is encoded by a nucleic acid sequence that may be operably linked to the heterologous nucleic acid sequence encoding a polypeptide of the invention. The signal peptide may also be a polypeptide tag, with an amino acid sequence comprising more than 30 amino acids.

The signal peptide acts as a polypeptide tag to promote selectively secretion of the polypeptide of the invention, e.g., a polypeptide tag that promotes the secretion of the polypeptide of the invention, i.e., promoting the secretion of a polypeptide hormone, such as but not limited to Exendin-4.

Accordingly, in embodiments, the polypeptide of the present invention comprises an N- terminal polypeptide, such as a signal peptide, selected from the group consisting of Alpha mating factor secretion signal, Alpha pre, Alpha pro, and functional homologues thereof. Accordingly, in embodiments, the polypeptide of the present invention further comprises an N-terminal polypeptide, such as a signal peptide, selected from the group consisting of signal peptides with an amino acid sequence of SEQ ID NOs: 41 , 43, 45, 47 and 48 and functional homologues thereof.

The signal peptide is typically selected from the group consisting of, but not limited to, signal sequences listed in table 2 below. In an embodiment wherein the heterologous polypeptide is part of a polypeptide comprising a signal sequence, the signal sequence is not part of the heterologous polypeptide as such and does thus not count when determining amino acid sequence identity as described herein.

Functional homologue

A functional homologue or functional variant of a protein/nucleic acid sequence as described herein is a protein/nucleic acid sequence with alterations in the genetic code, which retain its original and/or intended functionality. A functional homologue may be obtained by mutagenesis or may be a natural occurring variant from the same or other species. The functional homologue should have a remaining functionality of at least 50%, such as 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleic acid sequence. A functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality. A functional homologue can be a functional homologue of any one of the amino acid sequences shown in table 2, or a nucleic acid sequence encoding these sequences or amino acid, or nucleic acid sequences as disclosed in SEQ ID NOs: 1-100.

In embodiments, the heterologous nucleic acid sequences of the present disclosure further encodes an amino acid construct, comprising at least one secretion signal peptide which is operably linked to said one or more polypeptides.

In embodiments of the present disclosure, the genetically engineered cell further comprises a heterologous nucleic acid encoding a polypeptide hormone.

In embodiments, the polypeptide hormone is a GLP-1 analogue, such as but not limited to Exendin-4, GLP-11-37, GLP17-37, or GLP17-36, preferably, the GLP-1 analogue is Exendin-4 which has an amino acid sequence according to SEQ ID NO: 24.

In embodiments of the present disclosure, the polypeptide hormone is GLP-1.

In embodiments of the present disclosure, the polypeptide hormone is a GLP-1 analogue.

In embodiments of the present disclosure, the GLP-1 analogue is Exendin-4. In embodiments of the present disclosure, Exendin-4 has the amino acid sequence as defined by SEQ ID NO: 24 or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 24.

Another aspect of the present disclosure relates to a genetically engineered cell belonging to the genus of Saccharomyces, wherein the cell has been engineered to express and secrete a polypeptide hormone and has been further modified in such a way that at least two of a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the temperature sensitivity gene(s) BTS1 and/or REI1 are partially or fully inactivated in said cell, and/or c) the reduced oxidative stress tolerance gene(s) YAP1, SOD1, SOD2, CAD1 and/or MET18 is partially or fully inactivated in said cell.

In embodiments the THI6 and the BTS1 genes are inactivated in said cell. In embodiments the THI6 and the BTS1 genes are inactivated in said cell, and said cell expresses the GLP1 analogue Exendin-4, which has the amino acid sequence as defined by SEQ ID NO: 24 or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 24.

In embodiments the THI6, the URA3 and the BTS1 genes are inactivated in said cell, and said cell expresses the GLP1 analogue Exendin-4, which has the amino acid sequence as defined by SEQ ID NO: 24 or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 24.

Sequence identity

The term “nucleotide sequence encoding” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and generally represents the portion of a gene which encodes a certain polypeptide or protein. The term includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and doublestranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.

Within the scope of the present invention, also nucleic acid/polynucleotide and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs are comprised by those terms, that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a wildtype protein.

The term “sequence Identity of [a certain] %” in the context of two or more nucleic acid or amino acid sequences means that the two or more sequences have nucleotides or amino acid residues in common in the given percent when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (i.e., the sequences have at least 90 percent (%) identity).

Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g., http://www.ncbi.nlm.nih.gov/BLAST/). This definition also applies to the complement of a test sequence and to sequences that have deletions and/or additions, as well as those that have substitutions. An example of an algorithm that is suitable for determining percent identity, sequence similarity and for alignment is the BLAST 2.2.20+ algorithm, which is described in Altschul et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ is used to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Examples of commonly used sequence alignment algorithms are

CLUSTAL Omega (http://www.ebi.ac.uk/Tool s/msa/clustalo/),

EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss needle/),

MAFFT (http://mafft.cbrc.ip/alignment/server/), or

MUSCLE (htp://www.ebi.ac.uk/Tools/msa/muscle/) .

A nucleic acid sequence may be placed under the control of an inducible promoter, which is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors, bile acids and chemicals. Such promoters are referred to herein as “inducible” promoters, which allows timing of expression of the proteins used in the present invention. For Saccharomyces and other yeast cells, inducible promoters are known to those of skill in the art.

Expression of recombinant and/or heterologous genes in cells require that the codon content of the gene matches the codon usage of the cell, i.e., only genes comprising codons that are used by the specific organism are expressed, thus, recombinant and/or heterologous genes may need genetic modification before being capable of being expressed in a cell. The codon usage of common organisms is known to the person skilled in the art. Nevertheless, for proper expression of a recombinant and/or heterologous gene in a cell i.e., for the gene to be functional, the gene sequence may need to be optimized for expression in the specific cell, i.e., codon optimized.

As used herein, “codon optimization” refers to the process of optimizing the DNA sequence of a gene towards a specific host cell in order to improve expression of a gene of interest and increase the translational efficiency of a gene of interest by accommodating codon bias of the host organism. An example could be optimizing a human gene for expression in yeast. In general, as is known to the skilled person, when a gene is codon optimized, the codon optimization does not change the encoded amino acid sequence, but only relates to changes in the nucleic acid sequence, which does not reflect in changes in the amino acid sequence of the encoded polypeptide/protein.

The term "functional gene" as used herein, refers to a nucleic acid molecule comprising a nucleotide sequence which encodes a protein or polypeptide, and which also contains regulatory sequences operably linked to said protein-coding nucleotide sequence such that the nucleotide sequence which encodes the protein or polypeptide can be expressed in/by the microbial cell bearing said functional gene. Thus, when cultivated at conditions that are permissive for the expression of the functional gene, said functional gene is expressed, and the microbial cell expressing said functional gene typically comprises the protein or polypeptide that is encoded by the protein coding region of the functional gene.

Fully or partially inactivated genes

In the present context it is contemplated that various methods known to the skilled person can be used to achieve the genetically engineered cells with partially or fully inactivated genes disclosed herein.

These methods include but are not limited to deletion or knock-out of the genes in the yeast genome, deletion of the TATA-box associated with expression of the genes, insertion of stop codons or other terminator sequences, insertion of sequences into the promoter or protomer region of the genes, that disrupt promoter function. Examples of DNA/RNA primers and guide RNA molecules which can be used to obtain a genetically engineered cell of the present invention is provided in table 2, and SEQ ID NOs: 50-100.

In embodiments, the cell genes, such as, but not limited to BTS1 and THI6, are partially or fully inactivated by deletion of the genetic sequences in the yeast genome.

In embodiments, the cell genes are partially or fully inactivated by knock-out of the genetic sequences in the yeast genome.

Another approach for partially or fully inactivating the genes of the yeast is by gene knock-in, where the target gene(s) are replaced by another genetic sequence (see for example Steidler et al., 2003, Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10, Nat. Biotechnol.). An advantage of using gene knock-in based methods is that in cases where the knock-in mutant acquires a wild-type target gene and reverts back to the wild-type genotype, wherein the nucleic acid sequence inserted with the knock-in method is removed in the process. Thereby, any transgene(s) or heterologous genetic sequence inserted in the knock-in method are automatically removed during reversion to wild type genotype. Accordingly, in embodiments the full or partial inactivation of genes of the present invention is done by knock-in of one or more recombinant nucleic acid sequences. In that regard, in embodiments one or more of the genes BTS1, REI1, THI2, THI6, YAP1, CAD1, SOD1, SOD2 and MET18, are replaced with one or more nucleic acid sequences encoding one or more polypeptides of the present invention, such as a polypeptide hormone e.g., Exendin-4.

For the purposes of this disclosure, the term ‘partially inactivated’, refers to the modification or alteration of a gene or a segment of DNA in such a way that the expression, functionality, or activity of the gene is reduced or impaired but not completely eliminated. A gene can be considered ‘partially inactivated’ when its normal activity or function is diminished by at least 10% but not more than 90%, as measured by suitable methods known in the art such as quantitative PCR, reporter gene assays, or enzymatic activity assays. The degree of inactivation may be varied depending on the intended application and the desired level of gene product or activity. In most applications of the genetically engineered cell as disclosed herein, the genes referred to herein as being partially or fully inactivated are fully inactivated.

Thus, in embodiments, the target gene(s) is/are replaced by a heterologous nucleic acid or nucleic acid construct encoding a polypeptide hormone, preferably a GLP-1 analogue.

In preferred embodiments, the target gene(s) is/are replaced by a heterologous nucleic acid or nucleic acid construct encoding Exendin-4 peptide with an amino acid sequence according to SEQ ID NO: 24 or a functional homologue thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 24. Nucleic acid construct

The present invention also provides a nucleic acid construct comprising a recombinant nucleic acid sequence encoding one or more polypeptides of the invention, wherein the recombinant nucleic acid sequence may be flanked by regulatory elements that regulate the expression of said polypeptides.

Accordingly, a nucleic acid construct of the present invention comprises a nucleic acid sequence encoding a polypeptide of the present invention with a nucleic acid sequence according to SEQ ID NO: 23, 25, 27 and 29, or a functional homologue thereof with a sequence identity of at least 70 %, such as 80 %, such as 90 %, such as 95 % or such as at least 99 % towards SEQ ID NO: 23, 25, 27 or 29, or the reverse complement thereof. Preferably, the nucleic acid construct encodes Exendin-4, which is preferably encoded by a nucleic acid sequence which is at least 80 % identical to SEQ ID NO: 23.

Accordingly, a nucleic acid construct of the present invention comprises a nucleic acid sequence encoding a polypeptide of the present invention comprising or consisting of a nucleic acid sequence according to SEQ ID NO: 111 , 112, 113, 114, 115, 116, 117 or 118, or a functional homologue thereof with a sequence identity of at least 70 %, such as 80 %, such as 90 %, such as 95 % or such as at least 99 % towards SEQ ID NO: 111 , 112, 113, 114, 115, 116, 117 or 118, or the reverse complement thereof.

In embodiments, the heterologous nucleic acid sequence further comprises a nucleic acid sequence selected from SEQ ID NO: 40, 42, 44 and 46, which encodes a secretion signal peptide. Accordingly, in embodiments, the signal peptide is selected from polypeptides with an amino acid sequence according to any one of SEQ ID NOs: 41 , 43, 45, 47 and 48.

In addition, said nucleic acid construct may also comprise a non-coding nucleic acid sequence regulating the expression of said polypeptide.

A nucleic acid construct within the present context is an artificial construct comprising a nucleic acid insert that is integrated into or borne by a vector. The vector can be delivered via transformation/transfection to a host cell by for example physical, chemical, or viral methods and allow the nucleic acid inserts to be replicated or expressed in the host cell. Such methods for delivering a vector into a host cell are well known to the skilled person. Thus, the present disclosure also relates to a nucleic acid construct wherein expression of the one or more heterologous nucleic acids as defined herein are operably linked to one or more control sequences.

The term “operably linked” as used herein, shall mean a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence or a functional linkage between two or more polypeptides, i.e., a signal peptide and a polypeptide hormone.

The term "promoter" designates DNA sequences which usually “precede” a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA, non-limiting examples are promoter sequences according to any one of SEQ ID NOs: 31-36. “Regulator” DNA sequences, also usually “upstream” of (i.e., preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as “promoter/regulator” or “control” DNA sequence, these sequences which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual expression) of a gene will occur. DNA sequences which “follow” a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription “terminator” sequences, nonlimiting examples are terminator sequences according to any one of SEQ ID NOs: 37-39.

The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG). A coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.

As used herein, the terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary amino acid sequence thereof. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.

A recombinant nucleic acid sequence may be a coding DNA sequence e.g., a gene, or noncoding DNA sequence e.g., a regulatory DNA, such as a promoter sequence. A nucleic acid sequence comprising both the coding and non-coding sequences may also be referred to as a recombinant nucleic acid sequence.

In the present context, the term “recombinant nucleic acid sequence” or “recombinant gene/nucleic acid/nucleotide sequence/DNA encoding” are used interchangeably and intended to mean an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e. a promoter sequence.

In embodiments of the invention, the nucleic acid sequence encoding the one or more polypeptides is optimized for expression in the specific yeast strain, preferably, a yeast strain of the Saccharomyces genus, such as but not limited to saccharomyces boulardii.

The recombinant nucleic acid sequence may also be heterologous. As used herein “heterologous” refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism.

Thus, a nucleic acid construct provided by the present invention comprises a coding nucleic sequence, i.e., recombinant DNA sequence of a gene of interest, e.g., an Exendin-4 coding gene, and a non-coding regulatory nucleic acid sequence, e.g., a promoter or terminator DNA sequence, e.g., a promoter sequence derived from the TDH3 gene of Saccharomyces boulardii, such as a promoter with a nucleic acid sequence according to SEQ ID NO: 31.

In embodiments, these control sequences can be chosen as promoter elements and terminators.

The nucleic acid constructs can for example comprise one or more promoter or terminator sequences, wherein the one or more heterologous nucleic acids are placed under the control of one of said promoter or terminator sequences.

Preferably, the polypeptide encoding sequence is under the control of a promoter or terminator sequence selected from promotor sequences with a nucleic acid sequence as identified in Table 1 . Table 1. Selected promoter and terminator sequences

In further embodiments an expression vector and/or construct is integrated into the genome of the genetically engineered cell of the present invention. Integration of the nucleic acid construct of interest comprised in the construct (expression cassette) into the yeast genome can be achieved by conventional methods, known to the skilled person.

In embodiments, the one or more heterologous nucleic acid sequences are comprised in an expression vector.

In embodiments, the expression vector is integrated into the genome of the genetically engineered cell of the present invention. In other embodiments, the expression vector is integrated into a plasmid. In embodiments, the genetically engineered cell comprises a single genomic copy of the expression vector. In other embodiments, the genetically engineered cell comprises more than one genomic copy of the expression vector such as 2, 3, 4, 5, 6, or such as 10 genomic copies of the expression vector.

Genetically engineered cell

A " genetically engineered cell", is understood as a cell which has been transformed, engineered or is capable of transformation or engineering by a heterologous polynucleotide sequence. Preferably, the genetically engineered cell is a genetically engineered yeast cell. Appropriate yeast cells include yeast cells of the Saccharomyces genus. In preferred embodiments, the engineered yeast belongs to the Saccharomyces genus and is Saccharomyces boulardii. In embodiments, the genetically engineered cell of the present invention is a probiotic cell. In terms of the invention, a probiotic is to be understood as a live microorganism which when administered in adequate amounts confers a health benefit on the host. Often a probiotic cell is not integrated into the microbiome of the host in a permanent way but is instead excreted by the host over a period of time. An example of such a probiotic strain is Saccharomyces boulardii, which in example 5 and Table 7 is shown to be excreted from the host (mice) over a period of 10 days to 3 months, dependent on the host being treated with antibiotics or not.

The biocontainment strategy provided herein, allows for strict control of the colonization of the genetically engineered cells of the present disclosure, wherein the colonization is governed by either oxygen level, temperature and/or thiamine level. Such biocontainment strategy allows for partial colonization of the digestive system of the host by the genetically engineered cell, wherein the by the genetically engineered cell is excreted over time, thus not fully colonizing in the digestive system of the host, thus enhancing the safety profile of the genetically engineered cell. Thus, in embodiments, the yeast persists in the gastrointestinal tract of a host, such as a mammal, for less than 3 months, such as between 1 day and 3 months, such as less than 2 days, 4 days, 8 days, 10 days, 15 days, 25 days, 30 days, 40 days, 50 days, 60 days, 80 days, 90 days, 100 days or less than 130 days.

The term "genetically engineered" as used herein refers to the modification of the cell's genetic make-up using molecular biological methods. The modification of the cell's genetic make-up may include the transfer of genes within and/or across species boundaries, inserting, deleting, replacing and/or modifying nucleotides, triplets, genes, open reading frames, promoters, enhancers, terminators and other nucleotide sequences mediating and/or controlling gene expression. The modification of the cell's genetic make-up aims to generate a genetically modified organism possessing particularly desired properties. Genetically engineered cells can contain one or more genes that are not present in the native (not genetically engineered) form of the cell. Techniques for introducing exogenous nucleic acid molecules and/or inserting exogenous nucleic acid molecules (recombinant, heterologous) into a cell's hereditary information for inserting, deleting or altering the nucleotide sequence of a cell's genetic information are known to the skilled artisan. Genetically engineered cells can contain one or more genes that are present in the native form of the cell, wherein said genes are modified and re-introduced into the cell by artificial means. Genetically engineered cells can contain one or more genes that are present in the native cell, wherein said genes are fully or partially inactivated in the genetically engineered cell.

Genetically engineered cells can also be cells in which one or more genes have been deleted and therefore are absent compared to the native cell. Engineered cells can also be cells in which one or more genes have been substituted with one or more different gene(s), resulting in genetically engineered cell where the substituted gene(s) is/are absent when compared to the native cell and the different gene(s) are present instead or in increased numbers when compared to the native cell. The term "genetically engineered" also encompasses cells that contain a nucleic acid molecule being endogenous to the cell, and that has been modified without removing the nucleic acid molecule from the cell.

In embodiments, the present disclosure relates to a yeast belonging to the genus of Saccharomyces.

In embodiments, the present disclosure relates to a yeast belonging to the genus of Saccharomyces is selected from the list of species consisting of S. arboricolus, S. bayanus, S. bulderi, S. cariocanus, S. cariocus, S. cerevisiae, Saccharomyces boulardii, S. chevalieri, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, S. florentinus, S. fragilis, S. kudriavzevii, S. martiniae, S. mikatae, S. monacensis, S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, S. turicensis, S. unisporus, S. uvarum and S. zonatus.

In embodiments, the engineered yeast of the present invention is a yeast species belonging to the genus of Saccharomyces, selected from the group consisting of S. bayanus, S. cerevisiae, Saccharomyces boulardii, S. paradoxus, S. pastorianus, S. chevalieri, S. ellipsoideus, S. kudriavzevii, S. mikatae, S. cariocanus, S. norbensis, S. uvarum and S. eubayanys.

Saccharomyces boulardii is a marketed and FDA approved probiotic yeast and supporting evidence has shown that Saccharomyces boulardii is effective in complementing treatment against antibiotic-associated disorders, including Clostridium difficile infections, inflammatory bowel disease and other gastrointestinal disorders.

Saccharomyces boulardii is a variant of Saccharomyces cerevisiae that is referred to by the names Saccharomyces boulardii, Saccharomyces cerevisiae var. boulardii and Saccharomyces cerevisiae Hansen CBS 5926. Therefore, the term "Saccharomyces boulardii" as used herein is intended to refer to strains bearing any one of these three names. Accordingly, in embodiments, the engineered yeast of the present invention is Saccharomyces cerevisiae or a variant thereof, preferably Saccharomyces boulardii.

In preferred embodiments, the engineered yeast of the present invention is Saccharomyces boulardii.

Besides yeasts of the Saccharomyces genus, alternative yeast strains suitable to practice the present invention will be known to the skilled person, non-limiting examples of such are yeasts from the genera Debaryomyces, Hanseniaspora, Pichia, Meyerozyma, Torulaspora, Candida orthopsilosis, Candida tropicalis, Debaryomyces hansenii, Hanseniaspora osmophila, Kluyveromyces marxianus, Lachancea thermotolerans, Meyerozyma caribbica, Metschnikowia ziziphicola, Pichia fermentans, Pichia guilliermondii, Pichia kudriavzevii, Torulaspora delbrueckii and Yarrowia lipolytica.

In further embodiments, the genetically engineered cell is a non-pathogenic bacterial cell. Appropriate bacteria cells include bacterial cells of the Escherichia genus. In embodiments, the bacterial cell is a probiotic bacterial cell. In further embodiments, the engineered bacteria belong to the Escherichia genus and is E. coll Nissle 1917.

In embodiments, the bacteria are of the genus Escherichia.

In embodiments, the bacteria belong to the genus of Escherichia and is selected from the group of species consisting of E. albertii, E. coll, E. fergusonii, E. hermannii, E. marmotae, and E. vulneris.

In embodiments, the present disclosure relates to a bacterial cell selected from the group consisting of E. albertii, E. coll, E. fergusonii, and E. hermannii,

Several bacterial strains are used in production of dairy products and dietary supplements, such as Lactobacillus bulgaricus and Streptococcus thermophilus, but also strains of E. coll have gained impasse in the production of probiotic dietary supplements and in prophylactic treatment and in treatment of disease, the most well studied example of such an E. coll strain is E. coll Nissle 1917. Accordingly, in embodiments, the bacterial cell is E. coll Nissle 1917.

In further embodiments bacterial cell is a probiotic bacterial cell. In further embodiments bacterial cell is selected from the group consisting of Escherichia coli Nissle 1917, Escherichia coli Symbioflor G1/2, Escherichia coli Symbioflor G4/9, Escherichia coli Symbioflor G5, Escherichia coli Symbioflor G6/7 and Escherichia coli Symbioflor G8.

In embodiments, the invention also relates to a genetically engineered probiotic cell comprising a recombinant nucleic acid sequence encoding a cell-penetrating polypeptide selected from the group consisting of RRL Helix, Shuffle, Penetramax, and PN159, wherein the cell is E. coli Nissle 1917.

In further embodiments, the genetically engineered cell is E. coli Nissle 1917, wherein

I. one or more thiamine synthesis gene(s) are not partially or fully inactivated in said cell, and/or

II. one or more gene(s), which upon partial or full inactivation confer(s) temperature sensitivity are not partially or fully inactivated in said cell, and/or

III. one or more gene(s), which upon partial or full inactivation confer(s) a reduced oxidative stress tolerance, are not partially or fully inactivated in said cell.

In additional embodiments, the genetically engineered cell is E. coli Nissle 1917, which does not comprise partially or fully inactivated thiamine synthesis gene(s) THI2 and/or THI6, and/or partially or fully inactivated BTS1 and/or REI1 gene(s), and/or one or more partially or fully inactivated gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18.

Compositions

The present disclosure also relates to a composition comprising the genetically engineered cells as disclosed herein. Such a composition may further comprise one or more adjuvants and/or excipients. In embodiments, the composition of the present disclosure is provided as a pill, tablet, capsule or suppository. In further embodiments, the genetically engineered cells and/or composition of the present disclosure is used as a dietary supplement. In further embodiments, the genetically engineered cells and/or composition of the present disclosure is used as a food ingredient.

In the context of the present disclosure, the term "dietary supplement" is intended to mean any composition that comprises the genetically engineered cells as disclosed herein, which is intended to be ingested as an addition to the normal diet of a host or subject normal diet. A dietary supplement is a product that is intended to supplement the diet of a subject by the taking of for example a pill, capsule, tablet, powder or liquid. Dietary supplements classically include nutrient compounds such as for example vitamins, minerals, fiber, fatty acids, amino acids, plant pigments, omega 3 fatty acids or other compounds having a beneficial biological effect. The dietary supplements of the present disclosure may in further embodiments include one or more of the nutrient compounds mentioned above.

In the context of the present disclosure, the term "food ingredient" is intended to mean any composition that comprises the genetically engineered cells as disclosed herein, which is intended to be ingested in a food or drink. In example, a composition of the present invention may be used in fermentative processes such as beer brewing. Accordingly, a composition of the present invention comprising a yest cell as disclosed herein may be added to unfiltered beverages, such as but not limited to unfiltered beer. Additionally, a composition of the present invention may also be used in baking. When used as a food ingredient, the cell of the present invention may be used to express a polypeptide, either to obtain a food which is enriched with said polypeptide or the food may be used for the delivery of said cell to the gut of a subject, where it may express said polypeptide in the food and/or gut. In that regard, a composition of the present invention may be added to prepared food, or it may be used in the preparation of food. Accordingly, in embodiments, a yest cell of the present invention and/or compositions comprising said yest cell is for use as a food ingredient.

Preferably, a genetically engineered cell as disclosed herein is able to, at least partially, colonize in the digestive system of a host or a subject, for long enough to provide a health benefit to said host. Thus, in embodiments, the genetically engineered cells persist in the gastrointestinal tract of a host or subject for less than 3 months, such as between 1 day and 3 months or such as for at least 1 day, such as for example 2, 3, 4, 5, 6, or 7 days and no longer than 3 months. In additional embodiments, the genetically engineered cells persist in the gastrointestinal tract of a host or subject for at least 1 day, such as for example 2, 3, 4, 5, 6, or 7 days and no longer than for example 10, 14, 21 or 28 days.

In more embodiments, the genetically engineered cells persist in the gastrointestinal tract of a host or subject for at least 1 day, such as for example 2, 3, 4, 5, 6, or 7 days and no longer than for example 30, 60 or 90 days.

Furthermore, in embodiments, the composition of the present disclosure is formulated for delivery of the genetically engineered cells to digestive system of a host or subject. In further embodiments, the composition of the present disclosure is formulated for delivery of the genetically engineered cells to the gastrointestinal tract of a host or subject. In additional embodiments, the composition of the present disclosure is formulated for delivery of the genetically engineered cells to the small intestines of a host or subject. In preferred embodiments, the host or subject is a mammal, such as a human.

As shown in example 1 of the present disclosure, a genetically engineered cell of the present invention, may have a reduced ability to proliferate in a thiamine deprived environment. Thus, in a composition comprising a genetically engineered cell of the present invention, wherein the thiamine synthesis genes, THI2 and/or THI6 genes are fully or partially inactivated, the composition may preferably further comprise thiamine. Accordingly, in embodiments, the composition of the present invention comprises between 0.001 ug/mL and 1000 ug/mL thiamine. In further embodiments, the genetically engineered cell of the composition is lyophilized. In additional embodiments, the composition provided as a lyophilized powder in non-limiting examples, said lyophilized powder, may comprise between 1x10 ⁶ and 1x10 ¹² genetically engineered cells pr. g of lyophilized powder, such as about 1x10 ⁶ , 1x10 ⁷ , 1x10 ⁸ , 1x10 ⁹ , 1x10 ¹ °, 1x10 ¹¹ or 1x10 ¹² genetically engineered cells pr. g of lyophilized powder. In further embodiments, said lyophilized powder additionally comprises between 0.001 ug and 500 mg thiamine pr. g of lyophilized powder, such as about 0.001 ug, 0.1 ug, 1 ug, 10 ug, 100 ug, 1 mg, 10 mg, 100 mg or 1000 mg thiamine pr. g of lyophilized powder.

Probiotic microorganisms are usually provided as capsules comprising lyophilized microorganisms, in addition to further surfactants and/or excipients. The number of cells provided in each capsule varies but is usually in the range of 1x10 ⁸ and 1x10 ¹¹ cells pr capsule. Accordingly, in embodiments the composition of the present invention is provided in a capsule comprising between 1x10 ⁸ and 1x10 ¹¹ genetically engineered cells according to the present invention pr. capsule. In addition, the capsule may further comprise one or more further excipients and/or surfactants.

In embodiments, the present disclosure further relates to pharmaceutical compositions having as an active ingredient the genetically engineered cells as defined herein. Thus, in embodiments, the composition of the present invention is a pharmaceutical composition.

Pharmaceutical composition comprises in addition to the active ingredient, therapeutically inactive ingredients, such as a pharmaceutically acceptable or physiologically acceptable excipient, carrier and/or adjuvants, which are well-known to the person skilled in the art and may include, but are not limited to, solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g. solubility enhancing agents) colouring substances, fillers, preservatives, anti-oxidants, anti-microbial agents, viscosity adjusting agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, mucoadhesive substances, and the like. Examples of formulation strategies are well-known to the person skilled in the art.

In the present context a pharmaceutical composition is a mixture of ingredients suitable for administering to a subject that includes the genetically engineered cells as disclosed herein.

Use as functional food product

A cell, preferably of the Saccharomyces genus, according to the present invention can be used as functional food products, such as feed, food and/or food-supplements. In addition, a cell according to the present invention can also be used for production of polypeptide enriched functional food products. Consequently, said food, feed and/or food-supplement comprising a cell according to the present invention is equally disclosed. Such a food comprising a cell according to the present invention can be selected from the group consisting of soft-drinks, alcoholic and non-alcoholic beverages, yoghurt, food-bars etc.

By "functional food product" is meant, in the present context, a food product having a salubrious function, i.e. , having a beneficial effect on the health of man or an animal.

Accordingly, in embodiments, the cell of the present invention is used as a food ingredient, such as in an ingredient in a beverage and/or dairy product.

Further embodiments relate to the use of a cell and/or compositions according to the present invention as an ingredient in functional food.

Use as dietary supplements

The present invention also relates to a use of the genetically engineered cells and/or composition of the present invention as a dietary supplement and/or food ingredient. Thus, in an embodiment of the present invention, the genetically engineered cell is for use as a dietary supplement. In another embodiment of the present invention, the composition is for use as a food ingredient. As is shown in example 5, once administered to a host, the genetically engineered cell of the present invention does not affect the basal function of Saccharomyces boulardii, as the genetic modifications done to the strains do not affect the weight gain nor food intake of the host when compared to the basal Saccharomyces boulardii strain.

Saccharomyces boulardii is commonly used as a dietary supplement and is in example sold under the tradename SACCHAFLOR®. The genetically engineered cell and/or composition of the present invention is/are especially suitable as a dietary supplement due to its/their enhanced safety profile, which hinders cultivation of the cells in unintended environments. In addition, it was shown in example 4 that a host organism, provided with a composition according to the present invention comprising the genetically engineered cells of the invention, was cleared from the gut in a matter of days and it was highlighted that the biocontainment strains pose no noticeable fitness loss in the gastrointestinal tract of the host.

Accordingly, in embodiments, the cell and/or composition of the present invention is used as a dietary supplement, such as a weight reducing dietary supplement.

Further embodiments relate to a dietary supplement comprising a genetically engineered cell or composition as disclosed herein.

Further embodiments relate to the use of a cell and/or composition according to the present invention as an anti-inflammatory dietary supplement.

In embodiments, the present disclosure relates to the use of a cell and/or composition as disclosed herein as a probiotic.

In embodiments, the present disclosure relates to the use of a cell and/or composition as disclosed herein as a weight reducing dietary supplement.

In embodiments, the present disclosure relates to the use of a cell and/or composition as disclosed herein as a weight reducing dietary supplement.

In embodiments, the genetically engineered cells of the probiotic and/or dietary supplements as disclosed herein express and secrete a polypeptide hormone.

In embodiments, the polypeptide hormone secreted is GLP-1 or an analogue or variant thereof, such as but not limited to the GLP-1 analogue Exendin-4. In further embodiments, the polypeptide hormone secreted is Exendin-4. In embodiments, the invention relates to a weight loss product comprising a container, such as a tablet, capsule or suppository or another suitable pharmaceutical composition, comprising a genetically engineered cell or composition as disclosed herein.

In embodiments, the present invention also relates to a dietary supplement comprising a genetically engineered cell or composition as disclosed herein.

In embodiments, the present invention also relates to a consumable comprising a genetically engineered cell or composition as disclosed herein.

Use in modulation of gut microbiome

The gut microbiome is a complex composition of many different microorganisms that are entangled in the sense that they regulate each other through competition for the same nutrients and occupational space. The present invention relates to a use of the composition according to the present invention in the modulation of the gut microbiome of the host. In that regard, a composition of the present invention may be administered once or repeatedly to the host. Thus, in an embodiment, the composition of the present invention is administered once, and in another embodiment, said composition is administered repeatedly.

A microbiome is the community of microorganisms that can usually be found living together in any given habitat. In the present disclosure, microbiome refers to the community of microorganisms which is found in the gut of a host organism.

In general, the diversity of a host’s gut microbiome is described by a single identifier, namely the alpha diversity which is a measure of microbiome diversity applicable to a single sample. In general, a high alpha diversity is seen as beneficial for the host and many known medications, such as antibiotics, influences the alfa diversity in a negative way, i.e. , by reducing the alpha diversity due to the antibiotic effect. Thus, in embodiments, a cell and/or composition of the present invention may be for use in increasing the alpha diversity of a host microbiome following administration to said host. In additional embodiments, a cell of the present invention which expresses a cell penetrating peptide may be for use in increasing the alpha diversity of a host microbiome following administration to said host.

The alpha diversity is a guidance value, as upregulation of one beneficial microorganism might be beneficial, while upregulation of a pathogenic microorganism is generally unfavourable. The alpha diversity does not provide any indication as to the pathogenicity of the organisms but only describes the relative diversity.

To compare different microbiomes to each other, the beta diversity is used, which is a measure of similarity or dissimilarity of two communities, e.g., by a comparison of the microbiomes of different hosts.

Thus, in embodiments, a cell and/or composition of the present invention may be for use in increasing the beta diversity of a host microbiome following administration to said host.

As is shown in example 9, the cell penetrating peptides have different inhibitory on the growth of a selection of microbial cells. In embodiments, a cell of the present invention which expresses a cell penetrating peptide may be used for increasing the beta diversity of a host microbiome following administration to said host.

The microbiome of a host may be evaluated using any method known to the skilled person, examples of such are 16s/18s and Internal Transcribed Spacer (ITS)analysis of the host microbiome, which in brief are sequencing methods that can distinguish different genus from each other based on the specific sequence of the 16s rRNA gene, 18s rRNA gene or ITS region sequence of specific microorganisms, such as fungi or bacteria.

Furthermore, in embodiments, administration of a composition of the present invention to a host, enhances the beta diversity of said host’s bacterial microbiome.

In further embodiments, the alpha and/or beta diversity of a host’s bacterial microbiome is evaluated by 16s, 18s and/or ITS sequencing.

In addition, in embodiments, a composition of the present invention may enhance or reduce the alpha and/or beta diversity of a host fungal microbiome following administration to said host. In further embodiments, the alpha and/or beta diversity of said host fungal microbiome is evaluated by 16s, 18s and/or ITS sequencing.

An enhanced or reduced alpha and/or beta diversity of a host’s microbiome is evaluated as a comparison to the basal host microbiome as measured before administration of a composition of the present invention.

Examples 7-9 further indicates the use of cell penetrating peptides for modulating the permeability of epithelial tissue. Accordingly, in embodiments, a cell of the present invention, comprising a nucleic acid sequence encoding a cell penetrating peptide is for use as a permeation enhancer. In additional embodiments, said cell is for use in enhancing the paracellular permeability of the epithelial barrier.

Medical use

Clinical studies have revealed that Saccharomyces, and in specific Saccharomyces boulardii, has a clinical efficacy in treatment of several acute gastrointestinal conditions, including antibiotic-associated diarrhea (AAD), Clostridium difficile infection (CDI), acute diarrhea, enteral nutrition-related diarrhea, traveler’s diarrhea and Helicobacter pylori infection (Kelesidis et al., Efficacy and safety of the probiotic Saccharomyces boulardii for the prevention and therapy of gastrointestinal disorders, Therap Adv Gastroenterol. 2012 Mar; 5(2): 111-125).

Thus, the compositions, pharmaceutical compositions genetically engineered cell as disclosed herein are useful in the treatment of a variety of diseases as a microbiome-based therapeutic, such as, but not limited to acute gastrointestinal conditions, including antibiotic- associated diarrhea (AAD), Clostridium difficile infection (CDI), acute diarrhea, enteral nutrition-related diarrhea, traveler’s diarrhea and/or Helicobacter pylori infection.

As used herein Microbiome-based therapeutics is to be understood as a therapeutic process which is based on the delivery of a microorganism into the gastrointestinal tract or general digestive system of the host organism, wherein the microorganism produces and secretes one or more therapeutic molecules, such as for example one or more polypeptide hormones, that interact with the host organism.

Accordingly, embodiments relate to a use of the cell and/or compositions according to the present invention for use as a medicament.

Acute gastrointestinal conditions usually lead to enhanced inflammation in the host. The inflammation may be reduced by providing one or more probiotic cells which reduces the inflammatory state in said host. Accordingly, in embodiments, the cell and/or compositions according to the present invention is for use in the reduction of inflammation in a host. In further embodiments, the cell and/or compositions according to the present invention is for use in the treatment of acute gastrointestinal conditions, such as but not limited to antibiotic- associated diarrhea (AAD), Clostridium difficile infection (CDI), acute diarrhea, enteral nutrition-related diarrhea, traveler’s diarrhea and Helicobacter pylori infection.

In addition, a cell of the present invention may also express one or more recombinant therapeutic polypeptides. Such therapeutic polypeptides may also exert a therapeutic function. In example, the therapeutic polypeptide Exendin-4 may be used to treat obesity by reducing e.g., the food intake of the subject.

Accordingly, in embodiments, the cell and/or compositions according to the present invention is for use in the treatment of Alzheimer's disease, Parkinson's disease, Crohn's disease, type 2 diabetes, dementia, obesity and/or cardiovascular disease.

Thus, in embodiments, the present disclosure relates to the use of genetically engineered cells and/or compositions comprising said cell for use in the treatment of obesity.

In further embodiments, the present disclosure relates to a method of treatment of metabolic disorders comprising oral administration of an effective dose of a genetically engineered cell as disclosed herein.

In further embodiments, the present disclosure relates to a method of treatment of obesity comprising oral administration of an effective dose of a genetically engineered cell as disclosed herein.

In further embodiments, the present disclosure relates to a weight loss product comprising a container, such as a tablet, capsule or suppository, comprising a genetically engineered cell as disclosed herein, and optionally instructions for use.

Once provided to a host, the cell of the present invention may persist in the digestive system of the subject, such as a mammal, preferably in the gastrointestinal tract, for less than 3 months, such as between 1 day and 3 months, preferably, between 1 day and 20 days. General

It should be understood that any feature and/or aspect discussed above in connection with the genetically engineered cells according to the present disclosure apply by analogy to the methods described herein.

It should be understood that any feature and/or aspect discussed above in connection with the genetically engineered cells according to the present disclosure apply by analogy to each other.

It should be understood that the word “polypeptide” is used interchangeably with the word “peptide” and encompasses in example, oligopeptides, dipeptides, tripeptides, tetrapeptides, cyclic peptides and branched peptides, and other common alternative names known to the skilled person.

In the present disclosure the words “artificial sequence”, “synthetic construct” and “artificial construct” are used interchangeably.

The following Figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

SEQUENCES

The DNA sequences and peptide sequences of the THI2, THI6, BTS1, REI1, YAP1, CAD1, S0D1, S0D2 and MET18 genes found in several species of Saccharomyces are represented with the sequence listing and their corresponding sequences numbers are defined in the table 2 below.

Table 2 - overview of sequences used in the present disclosure.

ITEMS

1 . A genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell.

2. The genetically engineered cell according to item 1 , wherein the cell belongs to the species Saccharomyces boulardii.

3. The genetically engineered cell according to item 1 or 2, wherein THI6 and one or both of BTS1 and YAP1 are partially or fully inactivated in said cell.

4. The genetically engineered cell according to any of the preceding items, wherein the genetically engineered cell has a reduced viability and/or reduced ability to grow, in

I. a thiamine deprived environment, such as an environment with a thiamine level below 0.01 μg/ml, and

II. in an oxidated environment, such as in blood, and/or

III. in an environment with a temperature below 30 °C.

5. The genetically engineered cell according to any of the preceding items, further comprising one or more heterologous nucleic acids sequences encoding one or more polypeptides.

6. The genetically engineered cell according to item 5, wherein the polypeptide is a polypeptide hormone.

7. The genetically engineered cell according to item 6, wherein the polypeptide hormone is a GLP-1 analogue.

8. The genetically engineered cell according to item 5 or 6, wherein the polypeptide hormone is Exendin-4, GLP-I 1-37, GLP17-37, or GLP17-36, with an amino acid sequence according to SEQ ID NOs: 24, 26, 28 or 30, respectively, or a functional homologue of Exendin-4, GLP-11-37, GLP17-37, or GLP17-36, which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 24, 26, 28 or 30.

9. The genetically engineered cell according to any of the preceding items, wherein the cell comprises one or more heterologous nucleic acids sequences encoding one or more cell penetrating peptides.

10. The genetically engineered cell according to any of items 9, wherein the cell penetrating peptide (CPP) is selected from the list consisting of Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 comprising of consisting of an amino acid sequence according to SEQ ID NOs 102, 103, 104, 105, 106, 107,

108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108,

109, or 110.

11. The genetically engineered cell according to any one of items 9 or 10, wherein the cell penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110.

12. The genetically engineered cell according to any one of items 9-11 , wherein the cell penetrating peptide (CPP) is PN159 comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 110.

13. The genetically engineered cell according to any of items 5-12, wherein the heterologous nucleic acid encoding a polypeptide and/or a cell penetrating peptide further encodes an amino acid construct comprising at least one signal peptide which is operably linked to said one or more polypeptides and/or cell penetrating peptides, wherein said signal peptide is selected from the group consisting of polypeptides with an amino acid sequence as shown in SEQ ID NOs: 41 , 43, 45, 47 and 48.

14. The genetically engineered cell according to any of items 5-13, wherein the expression of said heterologous nucleic acids sequence(s) is under control of one or more promoter and/or terminator elements selected from the group consisting of promoter sequences of SEQ ID NOs: 31-36 and terminator sequences of SEQ ID NOs: 37-39.

15. A composition comprising the genetically engineered cell according to any of the preceding items.

16. The composition according to item 15, wherein the composition further comprises one or more adjuvants and/or excipients.

17. The composition according to item 15 or 16, wherein the composition is provided as a tablet, capsule or suppository.

18. Use of the genetically engineered cell according to items 1-14, or the composition according to any of items 15-17, as a dietary supplement and/or food ingredient.

19. A genetically engineered cell according to items 1-14, or the composition according to any of items 15-17, for use as a medicament.

20. A genetically engineered cell of the Saccharomyces genus, wherein i. the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and

II. one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell.

21. The genetically engineered cell according to item 20, wherein the cell belongs to the species Saccharomyces boulardii.

22. The genetically engineered cell according to item 20 or 21 , wherein the genetically engineered cell has a reduced viability and/or reduced ability to grow,

A. in an oxidative environment, such as in blood, and/or

B. in an environment with a temperature below 30 °C. 23. The genetically engineered cell according to any of items 20-22, further comprising one or more heterologous nucleic acids sequences encoding one or more polypeptides.

24. The genetically engineered cell according to item 23, wherein the polypeptide is a polypeptide hormone.

25. The genetically engineered cell according to item 24, wherein the polypeptide hormone is a GLP-1 analogue.

26. The genetically engineered cell according to item 24 or 25, wherein the polypeptide hormone is Exendin-4, GLP-I 1-37, GLP17-37, or GLP17-36, with an amino acid sequence according to SEQ ID NOs: 24, 26, 28 or 30, respectively, or a functional homologue of Exendin-4, GLP-11-37, GLP17-37, or GLP17-36, which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 24, 26, 28 or 30.

27. The genetically engineered cell according to any of items 20-26, wherein the cell comprises one or more heterologous nucleic acids sequences encoding one or more cell penetrating peptides.

28. The genetically engineered cell according to any of items 27, wherein the cell penetrating peptide (CPP) is selected from the list consisting of Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 comprising of consisting of an amino acid sequence according to SEQ ID NOs 102, 103, 104, 105, 106, 107,

108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108,

109, or 110.

29. The genetically engineered cell according to any one of items 27-28, wherein the cell penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110. The genetically engineered cell according to any one of items 27-29, wherein the cell penetrating peptide (CPP) is PN159 comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 110. The genetically engineered cell according to any of items 23-30, wherein the heterologous nucleic acid encoding a polypeptide and/or a cell penetrating peptide further encodes an amino acid construct comprising at least one signal peptide which is operably linked to said one or more polypeptides and/or cell penetrating peptides, wherein said signal peptide is selected from the group consisting of polypeptides with an amino acid sequence as shown in SEQ ID NOs: 41 , 43, 45, 47 and 48. The genetically engineered cell according to any of items 23-31 , wherein the expression of said heterologous nucleic acids sequence(s) is under control of one or more promoter and/or terminator elements selected from the group consisting of promoter sequences of SEQ ID NOs: 31-36 and terminator sequences of SEQ ID NOs: 37-39. A composition comprising the genetically engineered cell according to any of items 20-32. The composition according to item 33, wherein the composition further comprises one or more adjuvants and/or excipients. The composition according to item 33 or 34, wherein the composition is provided as a tablet, capsule or suppository. Use of the genetically engineered cell according to items 20-32, or the composition according to any of items 33-35, as a dietary supplement and/or food ingredient. A genetically engineered cell according to items 20-32, or the composition according to any of items 33-35, for use as a medicament. A genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and/or b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and/or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, S0D2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, and d) the cell comprises at least two of features a)-c), and wherein said cell comprises one or more heterologous nucleic acid sequences encoding GLP1 or an analogue thereof. A genetically engineered cell of the Saccharomyces genus, wherein a) the thiamine synthesis gene(s) THI2 and/or THI6 are partially or fully inactivated in said cell, and b) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, or c) one or more gene(s) selected from the group consisting of YAP1, CAD1, S0D1, S0D2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, and wherein said cell comprises one or more heterologous nucleic acid sequences encoding GLP1 or an analogue thereof. A genetically engineered cell of the Saccharomyces genus, wherein a) the gene(s) BTS1 and/or REI1, which upon partial or full inactivation confers cold sensitivity, are partially or fully inactivated in said cell, and b) one or more gene(s) selected from the group consisting of YAP1, CAD1, SOD1, SOD2 and MET18, which upon partial or full inactivation confers a reduced oxidative stress tolerance, is partially or fully inactivated in said cell, wherein said cell comprises one or more heterologous nucleic acids sequences encoding GLP1 or an analogue thereof. A genetically engineered cell according to any of items 38-40, wherein the GLP-1 or analogue thereof is GLP-11-37, GLP17-37, or GLP17-36, with an amino acid sequence according to SEQ ID NOs: 26, 28 or 30, respectively, or a functional homologue of GLP-1 _1-37 , GLP1 _7-37 , or GLP1 _7-36 , which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 26, 28 or 30. A genetically engineered cell according to any of items 38-41 , wherein the GLP-1 analogue is Exendin-4 with an amino acid sequence according to SEQ ID NO: 24, or a functional homologue of Exendin-4, which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 24. A genetically engineered cell according to any of items 38-42, wherein said cell further expresses a cell penetrating peptide. A genetically engineered cell according to any of items 38-43, wherein the cell penetrating peptide (CPP) is selected from the list consisting of Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 comprising of consisting of an amino acid sequence according to SEQ ID NOs 102, 103, 104, 105, 106, 107,

108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108,

109, or 110. The genetically engineered cell according to item 43 or 44, wherein the cell penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110. The genetically engineered cell according to any one of items 43-45, wherein the cell penetrating peptide (CPP) is PN159 comprising or consisting of an amino acid sequence according to SEQ ID NO: 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 110. The genetically engineered cell according to any of items 38-46, wherein the heterologous nucleic acid encoding a polypeptide and/or a cell penetrating peptide further encodes an amino acid construct comprising at least one signal peptide which is operably linked to said one or more polypeptides and/or cell penetrating peptides, wherein said signal peptide is selected from the group consisting of polypeptides with an amino acid sequence as shown in SEQ ID NOs: 41 , 43, 45, 47 and 48. The genetically engineered cell according to any of items 38-47, wherein the expression of said heterologous nucleic acids sequence(s) is under control of one or more promoter and/or terminator elements selected from the group consisting of promoter sequences of SEQ ID NOs: 31-36 and terminator sequences of SEQ ID NOs: 37-39. A genetically engineered cell according to any of items 43-48, wherein the cell penetrating peptide is fused to the GLP-1 or analogue thereof, such as via. a linker. A genetically engineered cell according to any of items 38-49, wherein the cell further expresses at least one signal peptide which is operably linked to the GLP1 or analogue thereof according to any of items 38-42, said cell penetrating peptide according to item 43-46, or said fusion product according to item 49. The genetically engineered cell according to any of items 38-50, wherein the expression of said GLP1 or analogue thereof as defined in items 38-42, said cell penetrating peptide as defined in items 43-46, said signalling peptide as defined in item 47 and/or said fusion product as defined in item 49, is under control of one or more promoter and/or terminator elements selected from the group consisting of promoter sequences of SEQ ID NOs: 31-36 and terminator sequences of SEQ ID NOs: 37-39. A composition comprising the genetically engineered cell according to any of items 38-51. The composition according to claim 52, wherein the composition further comprises one or more adjuvants and/or excipients. A genetically engineered cell according to any of items 38-51 , or a composition according to any of items 52 or 53, for use as a medicament. A genetically engineered cell according to any of items 38-51 , or a composition according to any of items 52 or 53, for use as a dietary supplement and/or food ingredient. A method of treatment of metabolic disorders comprising oral administration of an effective dose of a genetically engineered cell according to any of items 38-51. A method of treatment of obesity comprising oral administration of an effective dose of a genetically engineered cell according to any of items 38-51 . A weight loss product comprising a container, such as a tablet, capsule or suppository, comprising a genetically engineered cell according to any of items 38- 51 , and optionally instructions for use. A dietary supplement comprising a composition according to any of items 52-53. A consumable comprising a genetically engineered cell according to any of items 38- 53. A genetically engineered cell, which expresses one or more heterologous cell penetrating peptides (CPP). The genetically engineered cell according to item 61 , wherein the cell belongs to the Saccharomyces genus. The genetically engineered cell according to items 61 or 62, wherein the cell belongs to the species Saccharomyces boulardii. The genetically engineered cell according to any of items 61-63, wherein the cell comprises one or more heterologous nucleic acids sequences encoding said one or more cell penetrating peptides. The genetically engineered cell according to any of items 61-64, wherein the cell penetrating peptide (CPP) is selected from the list consisting of Tat, R9, R8, pVEC, Penetratin, Penetramax, Shuffle, RRL helix, and PN159 comprising of consisting of an amino acid sequence according to SEQ ID NOs 102, 103, 104, 105, 106, 107,

108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108,

109, or 110. The genetically engineered cell according to any one of items 61-65, wherein the cell penetrating peptide (CPP) is selected from the group consisting of Penetramax, Shuffle, RRL helix, and PN159 comprising or consisting of an amino acid sequence according to SEQ ID NOs: 107, 108, 109, or 110, or functional homologues there of which amino acid sequence is at least 80 % identical to any one of SEQ ID NOs: 107, 108, 109, or 110. The genetically engineered cell according to any one of items 61-66, wherein the heterologous nucleic acid further encodes an amino acid construct comprising at least one signal peptide which is operably linked to said one or more cell penetrating peptides, wherein said signal peptide is selected from the group consisting of polypeptides with an amino acid sequence as shown in SEQ ID NOs: 41 , 43, 45, 47 and 48. The genetically engineered cell according to any one of items 61-67, wherein the expression of said heterologous nucleic acids sequence(s) is under control of one or more promoter and/or terminator elements selected from the group consisting of promoter sequences of SEQ ID NOs: 31-36 and terminator sequences of SEQ ID NOs: 37-39. A composition comprising the genetically engineered cell according to any of items 61-68. The composition according to item 69, wherein the composition further comprises one or more adjuvants and/or excipients. The composition according to item 69 or 70, wherein the composition is provided as a tablet, capsule or suppository. A genetically engineered cell according to items 61-68, or a composition according to any of items 69-71 , for use as a dietary supplement and/or food ingredient. A genetically engineered cell according to items 61-68, or a composition according to any of items 69-71 , for use in enhancing the permeabilization of epithelial cells in a mammal. A genetically engineered cell according to items 61-68, or a composition according to any of items 69-71 , for use as a medicament. A method of enhancing the permeability of intestinal epithelial in a mammal, comprising administration of a genetically engineered cell according to any of items 61-68, or a composition according to any of items 69-71 , to a mammal by oral administration. A method of pre-treatment of a subject, comprising the administration of an effective amount of a genetically engineered cell according to any of items 61-68, or a composition according to any of items 69-71 , wherein said administration increases the permeability of the intestinal epithelial in said patient.

EXAMPLES

Methods for evaluating biocontainment features

Plasmids and strain construction

Oligonucleotides and gBIocks were ordered from Integrated DNA Technologies. To generate the URA3 ^S81X (SblT), HIS3 ^G26X (Sb H’), and TRP1 ^p12X (SbT’) disrupted strains, gBIocks with respectively gRNA were assembled with pCfB2312 (Cas9-KanMX) and transformed with their respective donor primer (see Table 2).

To generate the thi2A, thi6Δ, snz1A, sno1A, reilA and bts1A strains, gBIocks from respectively gRNA were assembled with pCfB3050 (pCas9-(JRA3) and transformed with respectively donor primer. The Exendin-4 integration plasmid (pCfB2909-Exe4) was implemented as previous described.

Table 3 - Plasmids

All plasmid assemblies, as described in Table 3 were conducted with Gibson Assembly and transformed into One Shot® TOP10 Escherichia coli (Thermo Fisher Scientific). All E. coli were grown in lysogeny broth (LB) media containing 5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCI; (Sigma Aldrich) supplemented with 100 mg/L ampicillin sodium salt (Sigma Aldrich). LB agar plates contained 1 % agar (Sigma Aldrich).

Saccharomyces boulardii (S. cerevisiae ATCC® MYA796TM) was obtained from American Type Culture Collection, ATCC. The strains created in this study are described in Table 4.

Table 4 - Strain and genotypes

Saccharomyces boulardii transformations were performed via high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Genomic integrations cassettes were digested with the restriction enzyme Notl (FastDgiest Enzyme, Thermo ScientificTM) prior to transformation and transformed together with various helper plasmid and preexpressed Cas9 from pCfB2312. All transformations were incubated at 30 °C for 30 minutes and then heat-shocked in a water bath at 42°C for 60 minutes. All transformations followed a recovery step. The transformation tubes were microcentrifuge for 2 min at 3000 g. Pellets were resuspended in 500 μL of YPD media containing 10 g/L yeast extract, 20 g/L casein peptone and 20 g/L glucose (Sigma Aldrich) and incubated for 2 to 3 hours at 30 °C before being plated. All yeast transformations were plated on synthetic complete (SC) plates containing 1.7 g/L yeast nitrogen base without amino acids and ammonium sulphate (Sigma Aldrich), 1 g/L monosodium glutamate (Sigma Aldrich), 1.92 g/L Yeast Synthetic Drop-out Medium Supplements without uracil (Sigma Aldrich) and 200 mg/L geneticin (G418; Sigma Aldrich) at 37 °C. Colony-PCR using OneTaq (Thermo ScientificTM) was used to confirm the genomic integration. Primers flanking the integration were used to confirm the integration. Genomic DNA was extracted by boiling cells at 70 °C for 30 min in 20 mM NaOH. The strains were cured for pCfB2312 and helper plasmids after genome integration.

Real-time growth monitoring

Real-time OD600 measurements were obtained every 10 min for approximately 72 hours with microplate reader SynergyTM H1 BioTek with an initial OD600 of 0.05. The cultures were incubated into 200 μL minimal synthetic complete media (DELFT) containing 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4 x 7H2O, 20 g/L glucose, 2 mL/L trace metals solution, and 1 mL/L vitamins with or without thiamine and pyridoxine, and with or without 20 mg/L uracil, 20 g/L histidine and 20 mg/L tryptophan supplemented. CELLSTAR® 96 well cell culture plate (Greiner Bio-One) with an air-penetrable lid (Breathe-Easy, Diversified Biotech) was used for all cultivation. pH was adjusted with 1 M HCI to 3, 4, 5 and 6 for the respective experiment. Cultivation was performed with continuous double orbital shaking of 548 cycles per minute (CPM) at 37°C and 0 %, 0.1 %, 1 % or 21 % oxygen. Thiamine dose-response experiment

The cultures were incubated into 200 μL SC media without thiamine for 48 hours with an initial OD600 of 0.05. The media was supplemented with either 0, 0.001 , 0.01 , 0.1 , 1 , or 400 μg/mL thiamine hydrochloride (Sigma Aldrich) and 20 mg/L uracil.

Escape rate experiment

The SblT and SbU'+thi 6Δ strains were incubated in 20 mL YPD for 72 hours. The cultures were incubated in a 250 mL shake flask, with an initial OD600 of 0.05. The culture was spun down and diluted in MilliQ water to OD600 10. A serial dilution from OD600 10 to 0.00001 was generated. 100 μL of the undiluted and 5 μL from each dilution was plated on SC plates with and without thiamine supplemented (Figure 3). The plates were incubated at 37 °C for 72 hours. Cell mass from the undiluted 5 μL spotting was spread out on SC plates with and without thiamine supplemented (Figure 3).

Cold exposure experiment

The SbU-, SbU-+rei1Δ, SbU-+bts1Δ A and SbU-+bts1Δ A+thi/6Δ strains were incubated in 2.6 mL YPD in a 24-deep well plate (Axygen®, VWR) with a sandwich cover (Enzyscreen) and with an initial OD600 of 0.05. The plates were incubated at 15, 20 and 37 °C for a maximum of 120 hours. OD600 was measured at 0, 8, 24, 32, 48, 72, 96, and 120 hours.

ELISA

The Exendin-4 producing Saccharomyces boulardii strains were incubated in 2 mL DELFT medium supplemented with 20 mg/L uracil in a 24-deep well plate (Axygen®, VWR) with a sandwich cover (Enzyscreen). The cultures had an initial OD600 of 0.05 and were performed with continuous shaking at 250 RPM at 37 °C. All cultures were harvested after 24 and 48 hours. Cell cultures were spun down at 10,000 g for 10 min at 4 °C. Exendin-4 was quantified with Exendin-4 EIA (EK-070-94, Phoenix). The signals were detected by OD450 using a microplate reader SynergyTM H1 BioTek.

Animal experiment

All animal experiments were conducted according to the Danish guidelines for experimental animal welfare, and the study protocols were approved by the Danish Animal Experiment Inspectorate (license number 2020-15-0201-00405). The study was carried out in accordance with the ARRIVE guidelines. All in vivo experiments were conducted blinded on male C57BL/6 mice (6-8 weeks old; Taconic Bioscience). Unless otherwise stated, all mice were housed at room temperature on a 12-hour light/dark cycle and given ad libitum access -o water and standard chow (Safe Diets, A30). All mice were randomised according to body weight and were acclimated for at least one week prior to the first oral administration. Each animal study received a freshly prepared batch of Saccharomyces boulardii. Body weight and food intake were recorded once per week.

The mice were divided into four groups (n = 4), either receiving the Sb wild-type, SbU-, SBU- +bts1Δ, SbU'+thi 6Δ or SbU'+bts1Δ+thi6Δ strain. The mice were orally administered via intragastric gavage with -10 ⁸ CFU of the Saccharomyces boulardii strain in 100 μL of 1x PBS and 10 % glycerol. The mice were orally administered with Saccharomyces boulardii for five consecutive days, followed by a six-day washout. The drinking water was supplemented with an antibiotic cocktail containing 0.3 g/L ampicillin sodium salt, 0.3 g/L kanamycin sulfate, 0.3 g/L metronidazole, and 0.15 g/L vancomycin hydrochloride after the washout period. After five days of antibiotic treatment, the mice were orally administered with Saccharomyces boulardii in 100 gL of 1x PBS (containing 1 .0 g/L ampicillin sodium salt, 1.0 g/L kanamycin sulfate, 1.0 g/L metronidazole, and 1.0 g/L vancomycin hydrochloride) and 10 % glycerol for five consecutive days. The washout for the antibiotic-treated mice was monitored for 33 days.

The faeces were collected in pre-weighed 1.5 mL or 2.0 mL Eppendorf tubes containing 1 mL of 1x PBS and 50% glycerol and weighed again to determine the faecal weight. All sample preparation for assessing CFU numbers was kept on ice and followed the same practice. The faecal samples were homogenised by vortexed at -2400 rpm for 20 min. The samples were then spun down at 100 g for 30 seconds, followed by a dilution series, where 5 μL of each dilution was plated in duplicates or triplicates. The faecal samples were plated on SC supplemented with 20 mg/L uracil plates containing 100 mg/L ampicillin, 50 mg/L kanamycin, and 30 mg/L chloramphenicol (Sigma Aldrich).

Statistical testing

All statistical analysing were performed in RStudio. Unless otherwise noted, all data are presented as mean ± SEM. The statistical significance level was set at p < 0.05 and confirmed with either One-way or Tow-way ANOVA. For experiments with multiple testing, either a Tukey’s HSD post hoc test or Dunnett’s post hoc test were conducted.

Methods for evaluating cell penetrating peptides Cell penetrating peptides

All CPPs peptides (Tat, RRL helix, R9, Shuffle, R8, pVEC, Penetratin, Penetramax and PN159) were synthesized by Genscript. Stock peptide solutions were prepared to have a final concentration of 1 mM and were stored at -20 °C in aliquots. Working concentrations of the peptides were prepared in appropriate mammalian cell culture or bacterial growth media for each experiment.

Working peptide concentrations

Stock solutions of all the CPPs were prepared in sterile distilled water at a concentration of 1 mM. Treatment solutions were made up of Dulbecco’s Modified Eagle Medium (DMEM) without phenol red (Sigma-Aldrich). Final working concentrations of the peptides in the treatment solutions were as follows: 1 , 5, 10, 50, and 100 μM in cell cytotoxicity and barrier integrity assays. In addition to these concentrations, 0.1 and 0.01 pM concentrations were included for antimicrobial assays.

Cell culture and maintenance

Human intestinal epithelial Caco-2 cell line was purchased from ATCC (catalog number: HTB-37). Caco-2 cells were grown in DMEM cell culture medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 1% MEM Non-Essential Amino Acids (NEAA) (Gibco; Thermo Fisher Scientific, Inc.), and 1% penicillin-streptomycin (Thermo Fisher Scientific) in an incubator at 37 °C with 5% CO2.

Caco-2 cell viability and cytotoxicity assays

Caco-2 cells were seeded (6X10 ³ cells/well) in Corning™ Costar™ 96-Well, Cell Culture- Treated, Flat-Bottom Microplate and grown at 37 °C with 5% CO2 until reaching confluence. For alamarBlue cell viability staining, cells were exposed to treatments (DMEM + AlamarBlue (10 μL) + CPPs) at different concentrations and incubated for 2 hours at 37 °C with 5% CO2. Fluorescence was measured with microplate reader Synergy™ H1 BioTek; (excitation wavelength: 560 nm, emission wavelength: 590 nm) and a curve of Relative Fluorescence Units (RFU) against different concentrations of CPPs was plotted.

In the case of Incucyte® Cytotox Dye (Sartorius) staining, cells were exposed to treatments (DMEM + Cytotox Red dye + CPPs) at different concentrations and incubated for 2 hours at 37 °C with 5% CO2. The cells were then imaged using the Incucyte® Live-Cell analysis instrument. Cytotox Red dye dilution: The dye was diluted to a stock concentration of 100 pM in PBS. This was further diluted in full media to yield a final concentration of 250 nM before adding to the cells. Measurement of TEER of Caco-2 monolayers

Transepithelial electrical resistance (TEER) represents the barrier integrity and permeability of the Caco-2 monolayers. To measure TEER, cells were seeded and grown on 12-well cell culture inserts (0.4 μm, 1.1 cm, polyethylene terephthalate membrane, celIQART) for a period of 21 days. TEER was measured using Millicell® ERS-2 Voltohmeter (MERS00002), combined with a STX-04 electrode. Final TEER values were expressed relative to the surface area of the inserts ( ΩTotai - Ωinsert X cm ² ). TEER values were monitored every day from day 14 until day 21 , before, and after permeability experiments. On day 21 of differentiation, TEER values of the monolayers were 600 ± 50 Ω X cm ² (mean ± SD; n = 48).

FITC-dextran translocation assay

For Fluorescein Isothiocyanate (FITC)-Dextran permeability experiments, Caco-2 monolayers differentiated for 21 days were transferred to 12-well plates containing 1.5 mL phenol red free DMEM media (pH 7.4) in the basal compartments. For the treatment, medium in the apical compartment was replaced by 0.5 mL phenol red free DMEM medium containing different concentrations of CPPs, and the plates were incubated at 37 °C for 1 hour. Post treatment, inserts were transferred to a new 12-well plate containing 1 .5 mL fresh phenol red free DMEM and the apical medium was replaced by medium containing 1 mg/mL FITC-dextran 4 (Sigma- Aldrich). The incubation with the permeability marker lasted for 30 minutes. Samples were collected from the basolateral compartments post incubation and fluorescence was measured using multi-well fluorescent plate reader (Synergy; excitation wavelength: 490 nm, emission wavelength: 520 nm). The concentration of FITC-dextran in the samples was calculated by comparing the relative fluorescence values to the FITC-dextran standard curve. The apparent permeability coefficient (P _app , cm/s) was calculated according to equation 1. equation (1 )

Where dQ/dt is the drug permeation rate (μg/s); a is the surface area of the inserts (cell monolayer) (cm ² ); and Co is the initial concentration at the apical side (μg/mL).

Bacterial strains and growth conditions

Gut commensal microbial strains used in the present application (Lactobacillus gasseri DSM 20077, Latilactobacillus sakei ATCC 15521 , Clostridium bolteae DSM 29485, Bifidobacterium longum ATCC 15697, Bifidobacterium adolescentis DSM 20083, Escherichia coli Nissle 1917 (Mutaflor), Escherichia coli K12 MG 1655, Bacteroides thetaiotaomicron DSM 2079, Bacteroides vulgatus ATCC 8482, Saccharomyces boulardii ATCC MYA-796) were purchased from American Type Cell Culture (ATCC) and DSMZ, Germany. The growth and screening experiments were performed in modified Gifu Anaerobic Medium (mGAM) (HyServe GmbH & Co) as all the strains could grow well in this media. mGAM was pre-reduced for a minimum of 1 day in the anaerobic chamber (Coy laboratory products Inc.) before use.

Broth microdilution assay for determining antimicrobial effects

The antimicrobial effects of CPPs were determined by performing microbroth dilution method. The overnight cultures of the strains were diluted 100-fold in mGAM broth and 100 μL of the diluted cultured were distributed in the 96-well microplate. Next, 100 μL of CPPs in their respective concentrations were added to respective wells. Plates were incubated aerobically (Escherichia coll Nissle 1917, Escherichia coll K12, and Saccharomyces boulardii) or anaerobically (rest of the strains) at 37 °C for 24 hours with or without shaking, respectively. Reading of each plate was performed by measuring the optical density (OD) at 600 nm in a microplate reader (Synergy™ H1 BioTek). The final absorbance values were normalized and represented as values between 0 and 1 as a heat map, where 0 being full inhibition and 1 being no inhibition The MIC was determined as the minimum concentration of the CPPs at which no significant growth of the strain was observed as compared to medium only (blank).

Statistical analysis

For statistical analysis, Graph Pad Prism software (Graph Pad Software Inc., San Diego, CA, USA) and Microsoft Excel (Microsoft corporation, USA) were used. Data were presented as means ± standard deviation (SD). Analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test was applied to determine statistically significant difference (P<0.01 ) as compared to the controls.

Example 1 - Auxotrophy strains

Impact of constraining the probiotic yeast to become dependent on the exogenous supply of metabolites for growth and survival, accordingly, one nucleoside (uracil), two amino acids (histidine and tryptophan) and two vitamins (thiamine and pyridoxine) auxotrophic strains (Figure 1 ) were generated as described in the “Methods” section.

A stop codon was introduced in the in URA3 (uracil synthesis), SNO1 (pyridoxine metabolism) SNZ1 (pyridoxine synthesis), HIS3 (histidine synthesis) and TRP1 (tryptophan synthesis) genes, as these auxotrophic strains have previously been reported in Saccharomyces boulardii. No growth of the strains with URA3, HIS3, and TRP1 disrupted was observed unless uracil, histidine or tryptophan was supplemented to the growth medium, while strains with disrupted SNZ1 and/or SNO1 genes showed significant growth in absence of pyridoxine (Figure 1 ). However, the TRP1 ^p12X (SbT-) strain showed a metabolic burden also when media was supplemented with tryptophan (Figure 1 ).

Accordingly, the HIS3 ^G26X (SbH-) and URA3 ^S81X (SbU-) strains are promising nutritional auxotrophs compared to the SbT- strain. The SbU' strain was selected for further gene knockouts based on a previous report showing higher gene expression from UF?A3-marker plasmids.

Disruption of two other biosynthetic pathways in strains with uracil synthesis deficiency to determine potential additive effects. Knocking out either gene in the thiamine synthesis pathway, THI2 and THI6, resulted in strains that were unable to grow in media lacking thiamine (Figure 1 and 2).

For the pyridoxine synthesis pathway, however, knockouts of either individual or both the SN01 and SNZ1 genes had a milder pyridoxine auxotrophic effect than previously reported. Nonetheless, it is worth noting that in the case when both the SN01 and SNZ1 genes were deleted, the growth defect is more notable than single deletions for either gene alone.

To demonstrate that the growth defect resulted from the respective auxotrophies, we also cultured the various strains in media containing the corresponding nutrient supplements and observed similar OD ₆₀₀ reaching after 48 hours (Figure 1B).

To ensure that the knockouts demonstrated enough robustness for in vivo application in the human gastrointestinal tract, we sought to evaluate the auxotrophic mutants’ growth performance in minimal media with pH and oxygen conditions more representative of the gastrointestinal tract.

Next thiamine sensitivity in the strains was investigated to determine the minimum thiamine concentration required for growth by the thi2Δ and thi6Δ strains to circumvent the biocontainment. Neither of the knockouts showed growth defect at a concentration as low as 0.1 μg/mL (Figure 2). The thi6Δ strain appeared more sensitive to low thiamine concentrations than the thi2Δ strain. Finally, it was confirmed that from a 72-hour culture, we observed a 0 % escape rate for thi6Δ strain (Figure 3). Example 2 - Cold-sensitive strains

Another strategy to reduce genetically modified microorganisms from proliferating in specific environments is to make them sensitive to temperature changes as it constrains the engineered microorganism to only survive in a specific temperature range.

To investigate this strategy, the two genes REI1 and BTS1 were knocked out in the SbU' strain. As can be seen in Figure 4 knocking out either one of the genes resulted in strains (SbU'+rei1Δ and SbU'+bts1Δ) that exhibited growth defects at cold temperatures of 15 and 20 degrees C when compared to wild type. rei1Δ displayed a more pronounced growth defect, showing no growth at 15 °C after 120 hours. However, at 20 °C, it started slowly growing after 96 hours (Table 5 and Table 6). Both SbU'+rei1Δ and SbU'+bts1Δ strains were hypersensitive at 15 °C; however, the SbU'+rei1Δ strain also showed a growth defect at 37 °C (Table 5 and Table 6).

Table 5 Growth performance of the cold-sensitive strain SbU'+btslA at different temperature. The mean OD600 over time at different temperature.

^A SD: Standard deviation

^AA Sig: Significance, Two-way ANOVA, Dunnett’s post hoc test with SbU- as reference.

Table 6 Growth performance of the cold-sensitive strain SbU-+bts1 A+thi6Δ and SbU-+rei1Δ at different temperature. The mean OD600 over time at different temperature.

^A SD: Standard deviation

''''Sig: Significance, Two-way ANOVA, Dunnett’s post hoc test with SbU' as reference.

Furthermore, evaluating the two cold-sensitive strains in gut-like conditions, it was demonstrated that the SbU-+rei1Δ strain showed a more pronounced fitness cost in minimal media at different pH (Figure 5). The SbU-+rei1Δ strain was unable to grow at a pH lower than 4 after 72 hours, and the strain displayed an approximate 25 % slower doubling time, i.e., slower growth, at pH 4, 5, and 6 compared to the parental strain (SbU-).

The reilA strain also showed a significantly lower growth rate i.e., higher doubling time in anaerobic and microaerobic conditions compared to SbU- (Figure 9), while the SbU-+rei1Δ strain demonstrated only a slower growth rate i.e., higher doubling time in anaerobic conditions. The growth defect at anaerobic conditions was comparatively more pronounced for the SbU-+rei1Δ strain than for the SbU-+rei1Δ strain.

Example 3 - Oxidative stress sensitive strain

Another strategy to reduce genetically modified microorganisms from proliferating in specific environments is to make them sensitive to oxygen/oxidative stress, as it constrains the engineered microorganism to only survive in a specific anaerobic environment.

To investigate this strategy, the gene YAP1, encoding the Basic leucine zipper (bZIP) transcription factor was knocked out in the SbU' strain, creating the SbU-+rei1Δ strain. As can be seen in Figure 10 knocking out the YAP1 gene resulted in a strain (SbU-+yap1Δ A) that exhibited growth defects when exposed to light, when compared to the SbU' strain. Example 4 - Auxotrophy and cold-sensitive strain

A more robust biocontainment strain was engineered by introduction of a combination of the auxotrophic (thi/6Δ) and cold sensitive (bts1Δ) mutations into the SbU' strain creating the SbU'+thi/6Δ+bts1Δ A strain. Combining the gene knockouts of BTS1 and THI6 maintained the phenotypic effect of the individual knockouts, demonstrating the inability to grow without thiamine supplementation (Figure 6Δ) and slower growth in temperatures lower than 20 °C (Figure 6B). Furthermore, combining the BTS1 and THI6 gene knockouts increased the thiamine sensitivity, showing a significantly lower OD600 in 1 μg/mL after 48 hours (Figure 11).

Introducing gene deletions can also cause a metabolic burden on protein synthesis, which is not reflected in the growth rate, resulting in abnormal protein disruptions and accumulation. Therefore, to investigate if the combined bts1A and thi6Δ biocontainment strain can produce a similar peptide concentration as the parental strain (SbU-), the bts1Δ and thi6Δ biocontainment strain ( SbU'+thi/6Δ+bts1Δ) was further engineered to produce and secrete the GLP-1 receptor agonist Exendin-4. No significant difference in the production of Exendin- 4 was observed after 24 and 48 hours (Figure 7).

Example 5 - In vivo biocontainment of strains

To test the safety and viability of the biocontainment strains disclosed herein, said biocontainment strains were administered orally to mice having an intact gut microbiome for five successive days followed by six days of washout, as measured by fecal level of selected strains following repeated dosing of 10 ⁸ cells (Sb, SbU'+thi6Δ, SbU'+Bts1A or SbU' +bts1 A+thi6Δ) for five days to mice (Figure 8, paradigm 1 ). As shown in Table 7, Paradigm 1 , the wild type, single knock-out strains and double knock-out strains all colonize equally in mice with an intact microbiome. Additionally, none of the tested strains were detectable in the mice after the 6-day washout period (except for a single mouse receiving the bts1Δ strain (Table 7, paradigm 1), indicating that the knock-out mutations do not cause undesired colonization and proliferation of Saccharomyces boulardii in mice.

Table 7. % of mice with S. boulardii colonized following treatment paradigm 1 and 2.

* Limit of detection = 50 CFU/g

Furthermore, the biocontainment strains were evaluated in an antibiotic-treated mouse model designed for pre-clinical testing of yeast-based microbiome-based therapeutics and investigated if the strains pose any unwanted colonization and proliferation (engraftment) or reduced survival in the mice with reduced microbiota (paradigm 2). Accordingly, the mice were dosed with ~10 ⁸ cells (Sb, SbU'+thi6Δ, SbLT+BtslA or SbU'+bts1A+thi6Δ) daily for five successive days, followed by 34 days of washout following the initial dosing (Figure 8, paradigm 2). The antibiotic treated mice were orally administered with the biocontainment strains for five days. Wild-type, single and combined knockouts were demonstrated to also colonise equally in the mice with a reduced microbiota (Table 8). Although the Saccharomyces boulardii was washed out slower in the antibiotic-treated mice, no mice displayed any detectable levels 33 days after the last oral administration (Table 7 paradigm 2 and table 8 paradigm 2).

Table 8. Colonization of strains over time following treatment paradigm 1 and 2, represented as mean Logio CPU per gram faeces in conventional mice with intact microbiota.

Limit of detection ~ 10 ³ CFU/g To investigate if the newly generated strains posed any safety concerns to the mice health and well-being, body weight, food intake and behaviour were monitored throughout the whole study. No change in in body weight (table 9) or food intake (Table 10) was observed in the mice receiving the biocontainment strains, both during low (paradigm 1 ) and high (paradigm 2) colonisation of the strains. In addition, no abnormal behaviour was observed in the mice.

Table 9. Body weight of mice treated with biocontainment strains under paradigm 1 and 2.

Table 10. Accumulated food intake (g) of mice treated with biocontainment strains under paradigm 1 and 2.

Example 6 - Exendin-4 producing cells

In the present example, it is shown that the Saccharomyces boulardii strains of the present invention to produce similar levels of a therapeutic compound, exendin-4, compared to the wild type Saccharomyces boulardii. Saccharomyces boulardii strains comprising knockout of URA3 or URA3 and THI6 or URA3 and BTS1 or URA3, BTS1 and THI6 were tested for their ability to produce Exendin-4. As can be seen in Figure 6, all the knockout strains produced the same levels of the therapeutic compound Exendin-4, demonstrating that the biocontainment strategies do not affect the synthesis of therapeutic proteins (Figure 7).

Example 7 - Effects of CPPs on toxicity and viability of Caco-2 epithelial cells

First, the effects of different concentrations of different CPPs (Tat, RRL helix, R9, Shuffle, R8, pVEC, Penetratin, Penetramax and PN159) on cellular health of intestinal epithelial cells were analyzed to determine a concentration for each of the CPPs that has no impact on cellular health. To determine that, two types of assays were carried out using human epithelial Caco- 2 cell lines treated with CPPs: 1 ) Cytotox Red dye staining was performed to visually differentiate between dead and live cells, 2) AlamarBlue staining was carried out to quantify the percentage of live cells after CPP treatment. Following the treatment of Caco-2 cells with CPPs, we observed three types of cytotoxicity outcomes: no evident toxic effect, highly toxic at 5 pM concentrations or a dose-response effect (Figures 12A, B). The majority of the CPPs (Tat, R8, R9, pVEC, and Penetratin) did not exhibit an evident cytotoxic effect on the Caco-2 cells at any of the tested concentrations. RRL helix and PN159 were toxic at 5 pM and continued to increase in toxicity with higher concentrations in a dose-response manner. Shuffle and Penetramax exhibited significant toxicity only at the highest concentration tested (100 pM). Furthermore, the percentage of viable cells after CPP exposure (Figure 12C) were calculated using the AlamarBlue cell viability assay. Results from the AlamarBlue assay were concordant with the cytotox Red dye staining except that the CPPs RRL helix and PN159 seemed to have no significant toxic effect at 5 pM. From these assays, we determined a concentration for each CPP at which no major cytotoxicity or effects on cell viability were observed. Example 8 - Effects of CPPs on epithelial barrier integrity

After determining a concentration at which no effects were observed on cytotoxicity and cell viability for each of the CPPs, the effect of the CPPs on the barrier integrity of the epithelial monolayers was evaluated. To do this, we measured the 1 ) transepithelial electrical resistance (TEER), and 2) translocation of Fluorescein Isothiocyanate labelled dextran (FITC-dextran) (Figure 13A). If the CPPs can modulate the tight junctions of the epithelial monolayers, we expect a reduction in TEER as well as an increase in FITC-dextran translocation from apical to basolateral side after treatment with CPPs. Most of the tested CPPs caused reduction in the TEER values of the Caco-2 monolayers after 1 h treatment (Figure 13B). Amongst these, RRL helix, Penetratin, Shuffle, pVEC, Penetramax, and PN159 caused significant reduction in TEER values, indicating high permeability of the epithelial barrier. In agreement with this, the same CPPs except for pVEC and Penetratin showed the highest paracellular permeability (passage from apical to basolateral side) of FITC-dextran (Figure 13C). As RRL helix, Shuffle, Penetramax, and PN159 seem to be most effective in improving intestinal permeability, these CPPs were selected to further study their impact on gut commensal microbes.

It was observed that CPPs Tat, R8, R9, pVEC, and Penetratin did not have an apparent toxic effect on epithelial cells (Figure 12B), but at the same time, these CPPs did not exhibit paracellular permeability in the concentrations tested as seen in TEER and FITC-dextran assays collectively (Figures 13B,C). Tat and Penetratin are among the most studied CPPs, which have been employed as a vector to improve intracellular delivery of various drug compounds, including oligonucleotides and proteins. Besides that, both Tat and Penetratin have been shown to promote permeation through tight junction modulation. However, as demonstrated in example 8 and shown in figure 13 C demonstrated Tat and Penetratin had no permeabilizing effect in the Caco-2 monolayer setup at 100 pM. Oligoarginine peptides R8 and R9 have been shown to be effective tools at improving intestinal absorption of therapeutic peptides, without damaging the epithelial barrier integrity in rats. In addition, polyarginine (R5) was shown to improve paracellular permeability of the nasal epithelium. However, no reduction in TEER or elevated FITC-dextran translocation was observed in the case of R8 and R9 at 100 pM in the experiments, which is in accordance with the results from a previous study (Bocsik et al., 2019), where R8 did not have an influence on the barrier integrity of Caco-2 cells even at 100 pM. This discrepancy in observations could be due to the following reasons: 1 ) difference in concentrations of CPPs used, 2) differences in experimental setup (in vitro vs. in vivo), and 3) differences in CPPs administration (CPP-drug conjugate vs. co-administration). In contrast, RRL helix, Shuffle, Penetramax, and PN159 significantly increased the paracellular permeability of the epithelial barrier when tested in concentrations that did not impact cellular health in vitro (Figures 13B,C). The results from the cytotoxicity and cell viability assays were in concordance except for RRL helix and PN159 at 5 pM. This minor difference most likely has to do with the sensitivity of the assay. The reducing capacity of the cells (as measured by AlamarBlue) is not the same as membrane integrity (as measured by Cytotoxic Red). In the present example RRL helix significantly improved both the TEER and FITC- dextran translocation, indicating paracellular permeability of the Caco-2 monolayers at 5 pM. Isolated RRL helix has previously been shown to successfully improve insulin absorption and bioavailability in rats. Furthermore, it has been shown that both Shuffle and Penetramax could reduce the TEER of the Caco- 2 monolayers and improve paracellular permeability of FITC- dextran, thereby altering the epithelial barrier integrity at 60 pM, like the TEER and FITC-dextran data from our results (Figures 13B,C). Finally, PN159 caused the highest reduction in TEER values and a drastic increase in FITC-dextran translocation, thereby exhibiting the highest paracellular permeability at 5 pM. The results presented herein are comparable to the results reported by Bocsik et al in 2019, where PN159 was shown to significantly improve paracellular permeability of Caco-2 monolayers via tight junction modulation at 3 pM. Based on their potent effect on paracellular permeability and low toxicity, we chose RRL helix, Shuffle, Penetramax, and PN159 to further evaluate their effect on representative microbial species from the gut microbiome.

Example 9 - Antimicrobial activity of CPPs

The antimicrobial activity of the most effective CPPs was tested against 10 gut microbial strains. The microbes tested include species from the most abundant phyla in the gut microbiome: Bacteroidetes (Bacteroides vulgatus and Bacteroides thetaiotaomicron), Firmicutes (Lactobacillus gasseri, Latilactobacillus sake!, and Clostridium bolteae), Actinomicetya (Bifidobacterium longum, and Bifidobacterium adolescentis) and Proteobacteria (Escherichia coli Nissle 1917 and Escherichia coli K12). In addition, we included a strain of Saccharomyces boulardii to evaluate the effect on yeast. Strains were cultured in aerobic or anaerobic conditions in incremental concentrations of the CPPs (0, 0.01 , 0.1 , 1 , 5, 10, 50 and 100 pM). Growth was determined by measuring the optical density of the cultures 24 h post-treatment and minimal inhibitory concentrations (MICs) were calculated for the CPPs for all the strains (Table 11 ) and are represented in Figures 14B-E.

Table 11: Minimum inhibitory concentrations (MICs) of CPPs against gut commensal strains.

*No inhibition is abbreviated Nl

All four tested CPPs displayed a dose-dependent antimicrobial effect (Figure 14A). However, some strains were more sensitive than others, for instance the Firmicutes strains tested were on average more sensitive than the rest of the strains. Concentrations as low as 0.01 pM affected the growth of L. gasseri, L. sakei and C. bolteae of the CPPs tested. In contrast, the Bacteroidetes species tested were able to tolerate higher concentrations of the CPPs. B. thetaiotaomicron and B. vulgatuswere able to tolerate most CPP concentrations for RRL helix, Shuffle, and Penetramax but were totally inhibited by PN159 (MIC = 10 pM). PN159 showed the most inhibitory effect against almost all the tested strains, where some being inhibited at concentrations as low as 1 pM (Figure 14E). RRL helix exhibited total inhibition (Figure 14D) against half of the tested strains (MIC = 5-100 pM) and a partial inhibitory effect on the remaining strains. Shuffle and Penetramax showed only moderate total inhibitory effects (Figures 14B, C) on most of the tested strains. S. boulardii was the most resistant strain against all the four CPPs as its growth was not affected even at 100 pM by any of the CPPs tested.

Herein the antimicrobial effects of the four best CPPs from the presented permeability studies is presented and it was found that all the CPPs had an antimicrobial effect against most of the gut microbial strains used in this study. The strains L. gasseri, L. sakei, and C. bolteae seemed to be most affected by the CPPs as their growth was affected either partially by all the CPPs in concentrations as low as 0.01 pM, then followed by B. longum and B. adolescentis strains, which were mildly affected in the lowest concentrations but were totally inhibited at slightly higher concentrations (MICs = 1 and 10 pM respectively). It is evident from the results that Gram-positive strains are more susceptible to CPPs than Gramnegative strains. This might be because Gram-negative bacteria have an outer cell membrane, which makes them more resistant to antimicrobials, while Gram-positive bacteria lack this layer. S. boulardii was resistant to all the four CPPs even at the highest concentrations of 100 pM. Similarly, the antimicrobial effects of pVEC, Penetratin, and PN159 against Saccharomyces cerevisiae has been tested and no inhibition in concentrations up to 25 pM was found. These results indicate that the CPPs might be active only against bacteria but less or not active against yeast. However, this can only be concluded after testing these CPPs against multiple yeast strains. PN159, which had a strong permeability effect, exhibited the strongest antimicrobial effect, inhibiting the growth of most strains as shown in Figure 14E (MICs = 1-50 pM). It is the only CPP that significantly inhibited the growth of both B. vulgatus (MIC = 10 pM) and B. thetaiotaomicron (MIC =10 pM). Previous studies have shown that PN159 can inhibit pathogens like Pseudomonas aeruginosa and Staphylococcus aureus at concentrations lower than 50 pM and E. coll K12 at 25 pM, while as demonstrated in example 9, the growth inhibition was already seen at 5 pM. This variation could be due to the differences in media used, strains tested or the experimental setup.

PN159 significantly improved paracellular permeability of Caco-2 cells and showed no significant effect on cell viability at 5 pM. However, due to its strong antimicrobial effects at its effective permeation concentration (Figure 14E), it might not be ideal for use as a permeation enhancer. RRL helix showed total inhibition only against 1 strain in its effective permeation concentration (Figure 14D). At the same time, the CPP did not cause high permeability of FITC- dextran as compared to Shuffle, Penetramax, or PN159. Possibly, a high dose of this peptide is required to improve paracellular permeability, but that might be toxic to the epithelial cells and cause more damage to the microbiome. Finally, Shuffle and Penetramax showed significant improvement in paracellular permeability in the TEER and FITC-Dextran assays without having significant effects on cell toxicity and viability at 50 pM. Looking at both partial (Figure 14A) and total (MICs) inhibitory effects (Figures 14B,C) of these CPPs, they have a relatively smaller antimicrobial effect in comparison to PN159. Therefore, from the data of the present application, considering toxicity to host cells, permeation enhancement effects, and antimicrobial activity of tested CPPs, we suggest that CPPs Shuffle and Penetramax might be suitable to use as permeation enhancers for oral drug delivery through the paracellular route. Of note, the data in this application are from in vitro experiments, therefore it is important to evaluate these CPPs in pre-clinical or clinical studies for a better understanding of their effects on intestinal permeability and safety profiles. The in vitro trans-well membrane setup contains only a layer of differentiated epithelial cells and lacks essential factors or barriers of the intestine like the mucus layers or the gut microbiota. These components are essential for maintenance of the epithelial barrier integrity and to carry out absorption by the intestine. Furthermore, the anti-microbial activity was tested against only 10 representative gut commensal strains. In contrast, animals and humans have a much more complex Gl tract, which is under constant exposure to a multitude of stimuli and the epithelial surface is in close contact with the mucus layer and is inhabited by billions of microorganisms. Further research should focus on evaluating these CPPs in more complex in vitro models like gut- on-a-chip, where it would be possible to co-culture stable microbial communities with epithelial cells in anaerobic conditions. In this application, we have highlighted the importance of evaluating the effects of CPPs on both target human cells and commensal gut microbes. These in vitro data provide a basis for selection of CPPs for further characterization in vivo with a goal to identify CPPs that safely and effectively enhance absorption of oral therapeutics specifically via the paracellular route in addition to the already known transcellular route, with minimal impact on the gut microbiome.

Example 10 - Expression of the CPPs in E. coli Nissle 1917 and S. boulardii

As a first step E. coli Nissle 1917 and S. boulardii are engineered to express the cell penetrating peptides (CPPs) RRL helix, Shuffle, Penetramax, and PN159. The CPPs are cloned with different combinations of promoters and ribosome binding sites (RBS) for the initial evaluation. As a second step the growth or fitness cost of all the strains are monitored to make sure they don’t have a growth defect either due to toxicity or metabolic burden of expressing the CPPs. Next step is to evaluate the CPP expressing strains effect on epithelial barrier integrity in vitro by TEER and FITC-dextran, and drug translocation assays as described in previous examples. The strains that significantly reduce the TEER and increase FITC-dextran and drug translocation are considered the best candidate. Next, the effect of CPP expressing strains on toxicity and cell viability of epithelial cells are to be studied in vitro. The most effective and non-toxic strains are validated in vivo in mice.

For an in vivo study, mice are orally dosed with the CPP producing strains for a period of 1 week. In this period, colonization of the strains in the mice is monitored regularly. On the last day, FITC-dextran and/or a commercial peptide drug is orally administered, and blood samples are collected from the mice at different time points. These blood samples will be used to measure FITC-dextran and/or the drug (indicating increased absorption), inflammation biomarkers, lactate dehydrogenase (LDH) etc. to make sure there is increased absorption without harming the host.

Example 11 - In vivo viability of oxidative stress sensitive strain

The present examples aim to address the viability of the biocontainment strains in vivo, specifically strains wherein the YAP1 gene is deleted, following direct injection to the bloodstream of mice.

Methods

All animal experiments were conducted according to the Danish guidelines for experimental animal welfare, and the study protocols were approved by the Danish Animal Experiment Inspectorate (license number 2021-15-0201-00926). All in vivo experiments were conducted on male BALBc mice (10 weeks old; Taconic Bioscience). Unless otherwise stated, all mice were housed at room temperature on a 12-hour light/dark cycle and given ad libitum access to water and standard chow (Safe Diets, A30). All mice were randomised according to body weight and acclimated for two weeks prior to intravenous injection.

The mice were divided into two cohorts with three groups in each cohort (n = 5 - 10), either receiving the Sb wild-type, SbU yap1A or SbU bts1A thi6Δ yap1A strain. The mice were intravenous (iv) injected in the tail with ~10 ⁵ CFU of S. boulardii strains per gram mouse, followed by either a 7-day (cohort one) or 28-day (cohort two) washout. Tail blood was collected from all the mice in cohort one at time point 0, 15, 30, 60 and 180 min post iv injection.

The mice were euthanised by anaesthesia (25% hypnorm, 25% dormicum in sterile water, (0.01 mL/g mouse)) followed by blood collection from the vena cava, and cervical dislocation for cohort one, and euthanised by cervical dislocation for cohort two. All tissues were collected in pre-weighted GentleMACS C tubes containing 1 mL of 1x PBS and weighed again to determine the tissue weight followed by homogenisation. All samples were plated on YPD agar plates containing 100 mg/L ampicillin, 50 mg/L kanamycin, and 30 mg/L chloramphenicol (Sigma Aldrich).

Results

An experiment was designed to test how well an S. boulardii strain with YAP1 deleted, causing the strain to be sensitive to oxidative stress, would survive the circular system of a mouse after being iv injected in the tail (Figure 1A). A faster clearance in the blood of S. boulardii was observed in mice receiving SbU bts1 A thi6Δ yap1A strain (Figure 15B) compared to the Sb wild-type. One week post injection a significant lower level of SbU bts1 A thi6Δ yap1A strain was detected in kidney, liver, and spleen (Figure 15C). No significance difference was observed in body weight between the different groups (Figure 15D and Figure 15E). All S. boulardii strains were below detection level 28 days post injection.

Example 12 - Yeast strain engineered to secrete cell penetrating peptides

In the present example, Saccharomyces boulardii was engineered to secrete the CPPs screened in examples 7-9. These were characterized for their ability to improve the absorption of FITC-dextran 4, a fluorescent marker, in vitro and in vivo.

Methods

Engineering of the yeast strains

The genes responsible for encoding cell-penetrating peptides underwent codon optimization for yeast and were synthesized as gblocks by Integrated DNA Technologies (IDT). The gene design ensured that both the N and C terminals had a 30-base pair overlap with the amplified plasmid backbone. These genes were then incorporated into the plasmid backbone through Gibson assembly. Subsequently, the plasmids were introduced into OneShot TOP10 Escherichia coli via electroporation. To validate the genes and their sequences, colony PCR and Sanger sequencing were employed.

Before being introduced into the yeast strain, the integration plasmids were extracted from the TOP10 strains and subjected to Notl enzyme digestion. Yeast transformations were carried out in an uracil auxotroph S. boulardii strain using the high-efficiency LiAc/SS carrier DNA/PEG method. These transformations involved the use of helper plasmids and preexpressed Cas9 from pCfB2312. The yeast cells underwent a heat shock at 42°C for 60 minutes followed by a recovery step. After centrifugation of the transformation mixture for 2 minutes at 3000 X g, the resulting pellets were re-suspended in 500 μL of YPD broth and incubated for 2 - 3 hours. Subsequently, the cells were plated on YPD agar plates lacking uracil. Genomic integration was confirmed via colony PCR, utilizing primers flanking the integration site. Genomic DNA was extracted by subjecting the cells to boiling at 70°C for 30 minutes in 20 mM NaOH. The products of the colony PCR were subjected to agarose gel electrophoresis, where the presence of a single amplification band around the size of 2000 bp indicated successful integration at both chromosomal sites. Finally, the strains underwent a removal process for pCfB2312 and the helper plasmids post-genomic integration.

The final strains constructed were as follows: Sb RRL helix had a single chromosomal integration of the RRL helix gene while the other CPP strains had a double chromosomal integration of the CPP genes. This is because the Sb RRL helix strain had a growth defect compared to the control strain (data not shown) in the growth curve experiments aerobically and therefore it was not possible to go for double integrations.

In vitro Caco-2 permeability assay procedure

Once the yeast strains were constructed, we tested the effect of cell-free spent media from the strains on the permeability of FITC-dextran 4 across Caco-2 monolayers. For this assay, Caco-2 cells were first differentiated as described previously by Gel li et al, 2023. On the day of the assay, Caco-2 monolayers were washed twice with PBS and transferred to a new plate containing fresh phenol-red free DMEM. The apical compartments were filled with the cell-free spent media from different yeast strains (Sb empty, Sb RRL helix, Sb Penetramax, and Sb PN159) along with 1 mg/mL of FITC-dextran 4. Samples were collected from the basolateral compartments at different time points.

In vivo study design

The studies were conducted according to the Danish guidelines for experimental animal welfare, and the study protocols were approved by the Danish Animal Experiment Inspectorate. The study was carried out in accordance with the ARRIVE guidelines. All in vivo experiments were conducted on male C57BL/6NTac mice (6 weeks old; Taconic Bioscience).

The study was designed to have 4 groups in total, which includes PBS group (n=6), Sb empty (12 mice), SbPN159 (12 mice) and another PBS group without FITC-dextran 4 for making the standard curve (See figure 17).

The mice were orally administered via intragastric gavage with ~10 ^A 8 CFU of S. boulardii (CPP producing strains or empty strain) in 100 μL of 1x PBS and 20% glycerol or only 1x PBS and 20% glycerol (PBS groups). The researchers were blinded in all mouse experiments.

In vivo study procedure

On the day of the assay, mice were fasted for 4 hours followed by oral administration of FD- 4 (0.6 mg/g) solution (100 mg/mL in PBS) in a randomized order. After 4 hours of dosing, mice were anesthetized using 25% hypnorm, 25% dormicum in sterile water (0.01 mL/g mouse), followed by blood collection from the heart and cervical dislocation. Plasma was separated using BD Microtainer® tubes (centrifuged at 6000 X g for 2 minutes) containing a PST ^TM plasma separator gel and lithium heparin additive. The plasma samples were diluted 1 :1 in PBS and fluorescence was measured with microplate reader Synergy™ H1 BioTek; (excitation wavelength: 485 nm, emission wavelength: 528 nm). Plasma from PBS dosed mice was used to make the standard curve.

Results

The genetically engineered yeast cells expressing RRL helix, Shuffle, Penetramax or PN159 were first tested in vitro for the ability to enhance the permeability of cellular monolayers (see figure 16). The Sb PN159 strain caused a significant increase in permeability of FITC- dextran 4 across the monolayers in 60 minutes while the Sb RRL helix and Sb Penetramax achieved the same in 90 mins (Figure 16). These results support the findings presented in example 7-9, with the synthesized CPPs. Unexpectedly, Sb Shuffle did not cause a significant increase in permeability of the marker. Considering this data, Sb RRL helix, Sb Penetramax, and Sb PN159 strains were further characterized in vivo in mice.

In the first in vivo study, the three successful CPP producing strains Sb RRL helix, Sb Penetramax, and Sb PN159 were tested in vivo in mice (figure 18).

The results showed a clear indication of increase in the translocation of FITC-dextran 4 into the blood when compared to the Sb empty control strain. While the other CPP strains did not show an effect (Figure 18).

Therefore, only the Sb PN159 strain was tested again in the second in vivo study with an increased group size and period of administration of yeast. Sb PN159 showed a significant increase in translocation of FITC dextran into the bloodstream compared to both the PBS and Sb empty control groups after 10 days of oral administration of Sb PN159 (Figure 18). Accordingly, the present disclosure for the first time discloses an engineered yeast that is engineered to secrete cell penetrating peptides.