Browsing by Subject "CRISPR Cas-9"

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Open Access
Engineering of probiotic Saccharomyces boulardii as a host for living therapeutic applications
(2022-09) Kurt, Orhan Nedim
Therapeutic molecules or biologically active agents used in the treatments of disorders have been manufactured employing chemical synthesis methods with high costs and limited accessibility. Even though this approach to producing therapeutic molecules has enabled life-changing substances to be produced, there are still countless diseases waiting for a cure. Therefore, it would not be possible to meet the treatment needs via just conventional manufacturing approaches. Living therapeutics are programmed cells for the production and direct delivery of therapeutic molecules to the human body. Indeed, biologically engineered cells have been used for a long time to manufacture biomolecules in industrial settings. Also, there have been many studies that designed and programmed living organisms to cure diseases such as cancer, diabetes, inflammatory bowel syndrome, ulcer, and colitis. accharomyces boulardii CNCM I-745 is a yeast strain used as a probiotic for a very long time in humans. S. boulardii is the first and the only yeast strain approved to be used in human medicine so far. It has been shown that S.boulardii has many benefits to its host, including restoring the microbiome and competing with pathogens. Also, S.boulardii is a promising organism to be engineered and programmed as a living therapeutic factory due to its great compatibility with the human body and its metabolic features enabling us to manufacture complex molecules. Aiming for this, we created a TRP1 auxotroph S.boulardii strain using synthetic biology tools to eliminate the use of antibiotic resistance genes in the further steps since it would be problematic for the spread of antibiotic resistance. To do that, we employed the CRISPR CAS-9 system and a donor DNA harboring a stop codon to be inserted into the target gene (TRP1). After verifying the construction of the TRP1 auxotroph strain, we designed an episomal expression vector comprising a strong, constitutive promoter, extracellular signal sequence (Alpha sequence), and a terminator (CYC1). Next, we inserted the Intrinsic Factor protein (the key protein responsible for vitamin B12 absorption) sequence into the vector to recombinantly produce it and secrete it into the gut. However, according to our preliminary data, we could not observe an expression of the protein of interest, suggesting that the system needs further optimization and investigation to work properly. Considering the lack of auxotrophic yeast strains available to be engineered for therapeutic purposes, our engineered S.boulardii strain can be employed in yeast-based living therapeutics applications against different disorders waiting for their treatment.