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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I2 - Engineering Probiotic Yeast for Anti-Inflammatory Modulation via IL-10 Therapy

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Christian N. Pile

Undergraduate student
Duke University
Durham, North Carolina, United States

Primary Investigator(s)

Cameron M. Kim, PhD (he/him/his)

Assistant Professor of the Practice in Biomedical Engineering
Duke University
Durham, North Carolina, United States

Introduction: Inflammatory bowel disease (IBD) refers to disorders involving chronic inflammation of tissues in the digestive tract, affecting about 3 million adults in the US. Disease flare-ups can be severe with 23-45% of ulcerative colitis and 75% of Crohn's disease patients requiring surgical GI tract removal [1]. Current IBD treatments suppress the immune system via immunomodulators which heighten the risk of infections and cancer, making these therapies less than ideal. Moreover, while over-the-counter probiotics utilize naturally selected anti-inflammatory mechanisms for maintaining gut homeostasis, they lack specificity for pathological inflammation [2]. Consequently, the development of an engineered probiotic that detects inflammatory biomarkers and responds by producing anti-inflammatory cytokines such as Interleukin-10 (IL-10) could significantly enhance IBD therapy.

We modified two strains of yeast for probiotic therapy- Saccharomyces cerevisiae and Saccharomyces boulardii. Saccharomyces cerevisiae is a strain of non-pathogenic yeast which can maintain human gut health by regulating endogenous flora and the immune system but is more commonly used in fermentation within the food and wine industries. Saccharomyces boulardii is a strain of probiotic yeast which is naturally present in the human digestive system and marketed as a dietary supplement.

Ultimately, the goals of this project are to evaluate the ability of yeast to secrete human IL10 for applications within a “smart” probiotic system as well as ascertain whether S. cerevisiae and S. boulardii are equivalent strains to engineer as part of this system.

Materials and

Methods: 1) Synthesis of IL-10 and fluorescent protein constructs.
- First, a pDJHO98-yhIL-10 expression plasmid was assembled in E. coli using Gibson cloning. The signal peptide from alpha mating factor(𝛼MT) was added to a yeast codon optimized human IL-10 fragment and subsequently inserted into a pDJHO98 backbone. The addition of 𝛼MT facilitates extracellular secretion. This plasmid was then cloned into a vector containing GAL-1 promoter to induce expression in glucose non-repressing conditions. Additionally, uracil is constitutively produced in this plasmid.
ISA1132 and pDJH098 used in the mKate protein construct were donated by Dr. Nathan Crook at NC State University.

2) Sequencing of plasmids grown in E Coli to verify proper sequences were assembled
- This was carried out using Sanger sequencing to compare with the expected vector map.

3) Selection of uniform colonies
- Both S. cerevisiae and S. boulardii strains were cultured on uracil dropout plates. These plates select yeast which have been successfully transformed using the Zymo Frozen-EZ Yeast Transformation kit.
- Individual colonies were grown in synthetic complete -URA media at 37°C and 250 rpm for 48 hours. Subsequently, uniform colony streaks were performed for clonal isolation. This step along with the reminder of the experiment was performed in triplicates for robustness

4) High-throughput growth of S. cerevisiae vs. S. boulardii yeast for hIL-10 and promoter expression.
-Transformed yeast samples were incubated under different sugar conditions for 48 hours at 37°C in a high-throughput 96 deep-well plate.

Results, Conclusions, and Discussions: 1) Genetic background changes mKate expression between S. cerevisiae vs S. boulardii.
- S. cerevisiae expresses mKate2 at ~2x levels compared to S. boulardii from the same plasmid when normalized for cell count and across different sugar growth conditions. mKate expression in S. cerevisiae ranged from ~400000 to 600000 RFU compared to ~100000 to 220000 RFU in S. boulardii.
2) Engineered yeast strains produce detectable hIL-10 under different sugar conditions.
- hIL-10 titer was measured by ELISA, revealing only S. cerevisiae under 2% dextrose + 2% galactose and S. boulardii under 2% dextrose displayed considerable hIL-10 secretion of ~ 12 pg/ml and ~10 pg/ml respectively. All other media conditions had no detectable levels of hIL-10.

Conclusions & Discussion
S. cerevisiae exhibited higher expression levels compared to S. boulardii which provides strong evidence for differences in genetic background directly influencing protein expression. Thus, specific yeast strains may be more useful to achieve desired outcomes with more measurement necessary.
S. boulardii should be better adapted to low pH and body temperature of 37°C, which serves as a better human probiotic, compared to the relatively neutral pH and lower fermentation temperature of 30°C deemed optimal for S.cerevisiae. However, the results of our experiment are contradictory as they indicated that S. cerevisiae expressed higher levels of mKate2 under simulated gut microbiome conditions. In future experiments, using microfermenter systems (e.g., BioLector) would provide a more appropriate system to effectively mimic the human gut.
The experiment also showcases that hIL-10 can be secreted by S.cerevisiae and S.boulardii at 37°C and varying carbon sources. However, not all growth conditions were conducive to this protein secretion. Large variations among biological replicates make it difficult to ascertain the efficacy of either strain in secretion of this protein.
hIL-10 secreted levels are well below therapeutic levels (ng/ml titers) when produced in the human gut. In further investigation, hIL-10 expression could be increased by testing various promoters and plasmid constructs. Ultimately, incorporating a sensing system to detect inflammatory biomarkers such as hypochlorite and reactive oxygen species, complementary to the secretion system would facilitate the creation of a “smart” probiotic.

Acknowledgements (Optional): [1]Praveschotinunt, P., Duraj-Thatte, A. M., Gelfat, I., Bahl, F., Chou, D. B., & Joshi, N. S. (2019, December 6). Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nature News. https://www.nature.com/articles/s41467-019-13336-6

[2] Scott, B. M., Gutiérrez-Vázquez, C., Sanmarco, L. M., da Silva Pereira, J. A., Li, Z., Plasencia, A., Hewson, P., Cox, L. M., O’Brien, M., Chen, S. K., Moraes-Vieira, P. M., Chang, B. S. W., Peisajovich, S. G., & Quintana, F. J. (2021, June 28). Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nature News. https://www.nature.com/articles/s41591-021-01390-x