Evolutionary engineering of rapamycin-resistant yeast
Evolutionary engineering of rapamycin-resistant yeast
Dosyalar
Tarih
2022-12-02
Yazarlar
Esen, Ömer
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Graduate School
Özet
The budding yeast Saccharomyces cerevisiae, a unicellular eukaryotic microorganism, is widely used in many industrial processes such as baking, alcohol fermentation, biofuel and recombinant protein production as well as in basic research to understand the complex biological processes of advanced eukaryotic organisms, including humans due to its well-characterized genome and proteome, ease of growth and manipulation, as well as the similarity of its genes and pathways to higher organisms. Rapamycin is a macrolide compound that is produced by the bacterium Streptomyces hygroscopicus. In the medical field, rapamycin and its analogs are used to prevent organ transplant rejection, coat coronary stents and treat tumor cells. Many key metabolic pathways including cell growth and lifespan, protein synthesis and ribosome biogenesis, regulation of cell cycle and size, environmental stress response, nutrient uptake, starvation control, and autophagy in S. cerevisiae and also other higher eukaryotes are affected by rapamycin due to its inhibition of the target of rapamycin (TOR). Fkbp12 forms a complex with rapamycin that interacts with TOR resulting in its inhibition where the exact mechanism is still unknown. Fkbp12, immunophilin, is conserved in its structure and function among eukaryotes from yeasts to mammalians. Fpr1 found in S. cerevisiae is an orthologue of Fkbp12 found in humans. In this study, an inverse metabolic engineering strategy, evolutionary engineering was used to obtain rapamycin stress-resistant S. cerevisiae. Thus, serial batch cultivation of the S. cerevisiae CEN.PK113-7D reference strain under gradually increasing rapamycin stress was carried out. Before selection, a screening experiment was performed and 3 ng/ml rapamycin stress was determined as the initial stress level. During the selection process, the concentration of rapamycin in the medium was gradually increased from 3 ng/ml to 200 ng/ml over 61 daily passages or populations. From the final population, fifteen individual colonies were randomly selected which were named R1 to R15. Every individual (R1 to R15) was highly resistant to rapamycin stress in comparison to the reference strain (905). Afterward, R1, R3, R7, R12 and R14 were selected to continue for genetic stability test in which they were found to be genetically stable and their resistance to rapamycin was shown to be permanent. Genetically stable strains were tested by spot assay for their cross-resistance or sensitivity against various stress types, including 0.5 mM NiCl2, 2.5 mM CrCl3, 3 mM CoCl2, 17.5 mM MnCl2, 50 mM NH4Fe(SO4)2, 10 mM AlCl3, 20 mM CuCl2, 15 mM LiCl, 50 mM H3BO4, 100 µM AgNO3, 0.5 M NaCl, 15 mM caffeine, 4 mM vanillin, 200 ng/ml propolis, 1mM coniferyl aldehyde and 200 ng/ml cycloheximide. R12 was found to be the mutant with the highest number of cross-resistance and sensitivities: R12 strain was cross-resistant to CuCl2, NH4Fe(SO4)2, NaCl, coniferyl aldehyde, vanillin, cycloheximide, propolis; and sensitive to AlCl3, CoCl2, H3BO4 and AgNO3 in comparison to the reference strain (905). In order to determine the cell wall integrity of the rapamycin-resistant mutant (R12), lyticase susceptibility assay was performed. The result of this experiment showed that R12 resisted lyticase more than 905, under nonstress condition. On the other hand, under rapamycin stress, 905 resisted lyticase more than R12. Furthermore, the presence of rapamycin stress did not change the lyticase resistance of the evolved strain R12, in comparison to the nonstress condition. The chronological lifespan of rapamycin resistant strain (R12) was determined by using semi-quantitative and also quantitative chronological lifespan analysis. The result of the experiments correlated with each other in which R12 was found to have a shorter CLS, in comparison to 905. Comparative whole genome sequencing analysis of the rapamycin-resistant mutant (R12) revealed four single nucleotide variations (SNVs). These SNVs were located in four different genes. The precise functions of these genes in rapamycin response and resistance in yeast should be examined in greater detail in future studies. In conclusion, a rapamycin-stress-resistant and genetically stable S. cerevisiae strain (R12) was successfully obtained by using evolutionary engineering in this thesis study, and characterized at genomic and physiological levels. These results indicate that TOR pathway-related changes occurred in rapamycin-resistant mutant in order to overcome the high levels of rapamycin stress. However, in order to fully comprehend the molecular basis of rapamycin-resistance of R12, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Açıklama
Thesis (M.Sc.) -- İstanbul Technical University, Graduate School, 2022
Anahtar kelimeler
yeast,
maya,
antibiotics-macrolide,
antibiyotikler-makrolid