LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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ÖgeInvestigation of the effects of abrb and cody deletions on the bacilysin overproducer B. subtilis HWA strain(Graduate School, 2024-07-12)Bacillus subtilis is a gram-positive, rod-shaped bacterium and a highly studied model organism. It has a highly adaptable metabolism and diverse physiological states regulated according to environmental conditions. B. subtilis species go through sporulation to form endospores that can survive harsh conditions such as high temperatures, and UV radiation. They can also form biofilms, attach to plant roots or fungal hyphae, take up extracellular DNA by its natural competence, show surface motility, produce and secrete secondary metabolites. Approximately 4-5% of the B. subtilis genome codes for secondary metabolites, including antibiotic bacilysin. Bacilysin, the main focus of this study, is a non-ribosomal dipeptide by linking non-proteinogenic amino acid anticapsin and L-alanine. Bacilysin causes selective cell wall disruption against bacteria, fungi, and algae species, some of which are pathogenic. Bacilysin is a pleiotropic signaling molecule for B. subtilis cells as it affects diverse cellular functions such as sporulation, germination and outgrowth. Previous studies have shown that the absence of bacilysin can have negative effects on spore quality and germination. A bacilysin non-producer strain was more sensitive to heat, chemicals and lysozyme. Additionally, comparative transcriptome analysis of B. subtilis PY79 and a bacilysin non-producer strain revealed that some genes related to competence development and biofilm formation are also affected by bacilysin. In B. subtilis, bacilysin biosynthesis relies on the expression of the bacABCDEF operon and a monocistronic gene bacG. Bacilysin production is regulated according to both external and internal factors. It has been established that growth conditions such as medium contents, temperature, and pH affect bacilysin production level. At the transcriptional level, bacilysin biosynthesis is regulated mainly via two mechanisms: quorum sensing pathway and stringent response that occur through the direct-action of the positive transcriptional regulators, including ComA~P, Spo0A~P, and LutR as well as negative transcriptional regulators AbrB, CodY, and ScoC. Bacilysin has broad-range activity against bacteria, with heat stability up to 15 min at 100°C, and activity within the pH range of 1.4 to 12.0. These characteristics give bacilysin significant clinical importance and make it an effective alternative to traditional drugs and biocontrol agents. However, bacilysin is produced at low levels, cannot be extracted with organic solvents, and has a low isolation yield. In our group, the bacilysin production level was increased at 2.87- fold via editing the 5' untranslated region (5'UTR) of the bac operon using the CRISPR/Cas9 approach, thereby obtaining the bacilysin overproducing strain B. subtilis HWA. Subsequently, to further boost the bacilysin production level in the over-producing strain B. subtilis HWA, the aim of this study was to examine how production levels are affected by eliminating the global regulators AbrB and CodY. To achieve this, the mutant strains B. subtilis PY79-GT0A (ΔabrB::cat) and B. subtilis PY79-GT0C (unkU::spc ΔcodY) were constructed by transforming competent PY79 cells with chromosomal DNA from the abrB deleted mutant strain B. subtilis BAL 373 (trpC2 pheA1 ΔabrB::cat) and the codY deleted mutant strain B. subtilis TMH 307 (trpC2 unkU::spc ΔcodY), respectively. Similarly, the mutant strains B. subtilis HWA HWA-GTA (ΔabrB::cat) and B. subtilis HWA-GTC (unkU::spc ΔcodY) were constructed by transforming competent HWA cells. Furthermore, the codY-abrB double mutant strains B. subtilis PY79-GT0AC (ΔabrB::cat unkU::spc ΔcodY) and B. subtilis HWA HWA-GTAC (ΔabrB::cat unkU::spc ΔcodY) were constructed by transforming competent cells of GT0A and GTA with chromosomal DNA from B. subtilis TMH 307. Potential tryptophan and/or phenylalanine auxotrophic mutants were eliminated by restreaking on solid Spizizen Minimal Media. Bacilysin phenotypes of the selected mutants were first detected by transferring colonies via toothpicks onto bioassay plates using Staphylococcus aureus ATCC 9144. Subsequently, mutants were grown in PA media for 16-18 hours and the bacilysin levels in their culture fluids were detected via paper disk-diffusion bioassay. Results showed that abrB disruption in HWA and PY79 cells significantly increased bacilysin production, though each strain was affected differently based on its baseline bacilysin production level. The bacilysin level in HWA-GTA increased by 7.7% relative to parental HWA strain, while PY79-GT0A displayed a 21.8% bacilysin level relative to its parent PY79. Interestingly, while codY mutation alone did not significantly affect bacilysin activity in HWA or PY79, adding codY mutation to AbrB mutants of both strains caused further increases of 22.1% and 13.4% relative to HWA and HWA-GTA, respectively, and 25.7% and 3.2% relative to PY79 and PY79-GT0A, respectively. In a final attempt to further enhance bacilysin production, the scoC mutation as an additional negative regulator of the bac operon was combined with the abrB-codY double mutation strain. However, the constructed triple mutant strain could not grow in liquid media, demonstrating that simultaneous disruption of these three global regulators severely compromised growth abilities of B. subtilis cells. In summary, the findings of this thesis study are important to revealing that concurrent inactivation of AbrB and CodY, two key negative regulators of bacilysin biosynthesis, provides the potential for improving bacilysin production levels further, even in the high-producing strain HWA.
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ÖgeInvestigation of protonation state dependent conformational dynamics of the nucleotide binding domain of Hsp70 protein homolog DnaK via computational methods(Graduate School, 2022)70 kDa heat shock proteins (Hsp70) are a ubiquitious and well-conserved protein family with chaperone functions. They supervise protein folding process and assist in proper protein folding and renaturation by preventing partially folded or misfolded proteins from forming amorphous aggregates and amyloid fibrils. In terms of structure, Hsp70s are composed of approximately 45 kDa N-terminal nucleotide binding domain (NBD) with ATPase activity, 25 kDa C-terminal Substrate Binding Domain (SBD) with substrate peptide binding pocket. Also, there is a conserved hydrophobic linker segment connecting the 2 domains. Hsp70 proteins do not work alone. Rather, they need aid of some other proteins called "cochaperones" to work optimally. The two auxillary cochaperones required by the Hsp70s are the 40 kDa Hsp40s(J domain proteins) and nucleotide exchange factors (NEFs). Together with the cochaperones, Hsp70 proteins form 3-membered Hsp70 chaperone machinery. For Hsp70 chaperone system to carry out all of its functions, the NBD and SBD domains must communicate with each other during the functional cycle. This mutual signal transfer and crosstalk between the domains is an intricate example of "allosteric regulation". Basically, nucleotide status of the NBD exerts an effect on substrate affinity. Reciprocally, binding of a substrate protein to the SBD stimulates ATPase activity of the NBD. In ATP bound state of the NBD, the two domains are in docked conformation. On the other hand, in ADP bound or nucleotide free state of the NBD, these two domains are undocked and become independent of each other. In this undocked conformation, the two domains are connected to each other with the hydrophobic linker. The ATP-bound state of the NBD with docked NBD-SBD conformation binds and releases substrates at much higher rates than nucleotide free or ADP-bound state; thus, ATP-bound state of the Hsp70 is known as "low-affinity state" state with substrate binding pocket open. In contrast, when the NBD lacks any nucleotide or is bound by ADP, rate of substrate binding and release occur at way slower rates. This state of the Hsp70 proteins is called as "high-affinity state" with closed substrate binding pocket. Hence, Hsp70s shuttle between open and closed states. The hydrophobic linker is an undispensable component of the Hsp70 chaperone system. By functioning as a dynamic signal switch, the linker conveys messages in either direction, from NBD to SBD and from SBD to NBD, keeping both domains in touch. In literature, the linker, particularly the hydrophobic 388 VLLL 392 sequence, have been demostrated to be vital for both interdomain communication and dynamics of the ATPase domain. Likewise, there are many residues of the NBD playing key roles in the Hsp70 cycle, particularly in the mechanism of ATP hydrolysis. Especially, D8, K70, E171, D194, T199, and D201 residues of E.coli Hsp70 homolog DnaK and correspondants of these residues in other Hsp70 homologs have been given attention. In this study it was aimed to elucidate the conformational dynamics of the nucleotide binding ATPase domain (NBD) of E.coli DnaK in nucleotide free state by means of computational simulation techniques. First of all, so as to understand if protonation states of critical residues of interest have any effect on the opening-closure dynamics of the NBD in nucleotide free state, initially open and closed structures with different protonation states were prepared. By changing the protonation states of residues D194, D201, and H226 in distinct combinations, charge states +12, +13, and +14 were attained. Four protonation states, namely D194 protonated (194pr), D201 protonated (201pr), both D194 and D201 protonated (2pr), and both D194 and D201 unprotonated (2dep) were tested for both initially open and initially closed conformations. In each case, another residue of interest H226 was taken in protonated form. To broaden conformational space scanned and explore contribution of linker position to the dynamics of the NBD, positions of the linker were changed manually multiple times for each protonation state. According to our standard molecular dynamics (MD) simulation results (each 500 ns) of DnaK 1-392 construct of initially open conformations with these 4 protonation states, it was seen that protonation state can dramatically influence the tendency of initially open conformation towards closure. The cases in which D194 were unprotonated (2dep and 201pr) exhibited a tendency to close. Especially, 2dep became totally closed after 200 ns simulation period. On the other hand, 194pr had no tendency towards closure at all. On the other hand, all of the initially closed conformations for each of the 4 protonation states of the DnaK 1-392 retained their closed forms. Based on these simulation results, it can be deduced that the energy barrier between open and closed conformations was highest for 194pr and lowest for 2dep. In order to both decide on relative abundance of open - closed structures and circumvent sampling problems encountered during classical MD simulations, we carried out temperature replica exchange molecular dynamics (T-REMD) simulations for each protonation state. In addition to the 4 protonation states with protonated H226 residue investigated during MD simulations, 2 additional states, namely D194 protonated-H226 deprotonated (194prHID226) and D201 protonated-H226 deprotonated (201prHID226), were prepared by only removing the epsilon H atom of H226 from 194pr and 201pr cases, respectively. To elucidate whether the linker favors closed conformations, apart from these 6 different protonation cases with residues 1-392 of NBD, 6 further constructs were prepared by stripping the last 4 residues 389VLLL392 of the linker away from each of 1-392 construct. In total, 12 T-REMD simulations, 6 with 1-392 and 6 with 1-388 residues, and each with 300 ns-long were performed. Based on our T-REMD results, the most essential point to be understood is that closed conformations are much more favorable compared to open ones. Considering the ratios of open conformations, even the highest fractions of open conformations obtained in the case of 194pr388 did not exceed 25%. Looking at other cases, the percentage of open conformations can be as low as 2-3% for 201pr cases. If we look at 194pr cases, irrespective of the presence of last 4 residues of linker and protonation state of H226, open structures are most abundant in the cases of 194pr. Among 194pr and thereby all other protonation states-structures, 194pr388 (226 protonated) promotes, by far, the open structures most. Additionally, the role of the length of linker in favoring closed structures over open ones becomes more prominent when H226 is protonated with nearly 7% higher closed frames in 194pr392 than 194pr388. As opposed to 194pr, regardless of linker or protonation state of H226, 201pr cases are out and away the protonation states that exhibit lowest tendency towards opening with fraction of open structures no more than 5%. Unlike 194pr, there is no evident impact of linker on opening-closure behavior. Protonation state of H226 seemed not that important in either protonation state. In agreement with the standard MD simulations, 2dep was monitored to tend to have high number of closed frames. Indeed, 2dep comes after 201pr in terms of closed structure fractions. The contribution of the linker to the closure of the NBD was minor and seen only the last 200ns. As to 2pr states, the 2nd highest fraction of open frames, both in the presence and absence of 4 terminal residues of the linker, after 194pr were obtained. Another point of interest in this study was to scrutinize the pH dependence of DnaK ATPase domain. The active site of the ATPase domain comprises multiple charged amino acid residues; therefore, it can be expected from Hsp70 proteins to be susceptible to changes in pH conditions. To the best of our knowledge, only two experimental studies in early 2000's underlining pH dependent behavior of isolated DnaK NBD have been conducted thus far. On the other hand, no computational study has paid attention to change in the dynamics and activity of either full-length protein or NBD alone with respect to pH. Hence, which of the candidate residues in the active site are in protonated or deprotonated form around physiological pH remained elusive. To solve this mystery, thermodynamic integration (TI) simulations were performed, again for both DnaK 1-392 and DnaK 1-388. For TI simulations, for each of 12 protonation states used in the T-REMD, 2 initially closed structures alongside one open structure were picked from cluster analysis performed on the T-REMD trajectories. Additionally, pKa values of E171 and epsilon position of H226 were calculated in various protonation/deprotonation scenarios of D194 and D201. Regarding pKa values of epsilon H226, we inferred that pKa values were more or less the same around 9, indicating the fact that neither protonation states of active site aspartate and glutamate residues nor the lenght of the linker altered pKa values of H226. Protonation state of E171 is likely to depend on the protonation states of D194 and D201. When these aspartates were both deprotonated, E171 was protonated. D194, D201, and H226, on the other hand, must be in protonated states around physiological pH conditions. Consequently, according to our pKa values, we got +12 charge state, not +13 or +14. All in all, combining all atom MD simulations with REMD simulations and free energy TI simulations, the following questions were tried to be clarified in this thesis study: 1) Effect of protonation state on opening-closure abundance and dynamics of NBD 2)Whether the linker induces a change in the ratio of open to closed conformations. 3) pH dependence of the NBD through demonstration of the possible protonation/deprotonation status of critical residues 4) Whether linker promotes change in pKa of any of critical active site residues. At the end, we came to these conclusions. Dynamics of the DnaK NBD opening-closure were protonation state dependent. Intriguingly, protonation of 2 active site aspartate residues have opposite effect in such a way that protonation of D194 increases the abundance of open conformations, whereas D201 promotes more closed form of the NBD. In addition, at different charge states, there must be multiple protonation states in equilibrium. Regarding the linker, there was no direct indication of the linker being involved in opening/closure or pH dependence of the isolated DnaK NBD. Likewise, no apparent role of the linker in shifting the pKa of any critical residue in the active site.
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ÖgeExpression, purification and characterization of high-fidelity DNA polymerase(Graduate School, 2022)DNA polymerases found in all living cells discovered to date are the enzymes that synthesizes a new DNA strand complementary to template single stranded DNA. These enzymes do not only play a major role in the transmission of genetic information across generations during cell division, they also form the basis of Polymerase Chain Reaction (PCR), which is one of the most important in-vitro diagnostic techniques today. In addition to synthesis ability, DNA Polymerases may also have other properties including processivity, which is known as the ability of continuous polymerization, fidelity, which is known as the synthesis accuracy, and nucleotide selectivity. Thermostable DNA polymerase enzymes are mostly preferred in PCR-based studies because it is high importance that the stability of the enzymes used do not decrease depending on temperature. Taq DNA polymerase is the first discovered polymerase, which is a well-known enzyme used in a wide range of applications. Following the discovery of Taq DNA polymerase, the high-fidelity DNA polymerase was discovered in 1991 as a highly thermophilic DNA polymerase. Due to its high thermostability and proofreading properties, high-fidelity DNA polymerase is widely used in the applications that require high accuracy such as molecular cloning. High-fidelity DNA polymerase is an enzyme with a length of 775 amino acids and a molecular weight of about 90 kDa. This enzyme can perform 3'-5' exonuclease (proofreading) activity, which allows the addition of the correct nucleotides by removing the wrong nucleotides added to the structure during DNA synthesis. Due to this feature, it reduces the error rate during synthesis (1.3×10-6 mutations/base pairs/duplications), resulting in about 8 times less errors compared to Taq DNA polymerase. Many researchers have produced this protein by cloning it from Pyrococcus furiosus, a hyperthemophilic archaea, into different strains of Escherichia coli. The purification step is simplified by adding an affinity tag to the N- or C-terminus during the cloning. Based on these tags and various biophysical properties of the protein, purification protocols were created by affinity chromatography or ion exchange chromatography. In this study, we aimed to purify and characterize the high-fidelity DNA polymerase enzyme by taking the advantage of its thermal stability and 10X Polyhistidine-tag after bacterial production with high efficiency and low cost. For this purpose, commercially purchased pET16B. High-fidelity polymerase's plasmid DNA with a 10X Polyhistidine-tag at the N-terminus was used. The plasmid pET16B. High-fidelity polymerase was transformed into competent E. coli BL21(DE3) cells containing GroEL/GroES chaperonins to ensure soluble expression of the protein. In the first step of purification of High-fidelity DNA polymerase, which is a thermostable protein, all the folded proteins obtained from bacterial cells were heated and centrifuged to separate impurities with less thermal stability. High-fidelity DNA polymerase in soluble form was purified using IMAC affinity chromatography. The pure product was taken into a storage buffer containing 50% glycerol by filtration. The GroEL/GroES chaperonin system is a system that enables unfolded proteins with a molecular weight of 2-100 kDa to be folded in vitro and in vivo. Given that GroEL/GroES system can increase the folding of co-expressed recombinant proteins of different sizes by up to 70%, this system was employed in the production of High-fidelity DNA polymerase. However, while this system increases the amount of target protein, it can also increase the amount of impurities. Therefore, the purification of High-fidelity DNA polymerase was highly challenging. So that, various buffer compositions were used in order to optimize one step IMAC purification. Co-expression system was induced using IPTG for expression of pET16B. Expression of the polymerase regulated by the Lac operon. Since the growth temperature was chosen in the range of 12-20°C, where the metabolic rate of the cell and thus the growth rate was selected, the amount of protein folded by the chaperones was increased. Most of the impurities that increased with the target protein were eliminated with the 90°C heat treatment step. While heat sensitive proteins are eliminated from the environment, thermostable high-fidelity DNA polymerase enzyme, GroEL, and GroES proteins are still present. The separation of GroEL and GroES was achieved by applying IMAC affinity chromatography to increase the purity of the high-fidelity DNA polymerase enzyme. Purification results were analyzed by SDS-PAGE and immunoblotting methods. At the end of 500 ml bacterial production and purification process with three biological repetitions, high-fidelity DNA polymerase enzyme of similar quality with its commercial counterparts was produced, which can be used for a total of 60 000 PCR reactions with ~90% purity. The protein band on the SDS-PAGE gel was excised and analysed by peptide mapping using Liquid Chromatography-Mass Spectrometry (LC-MS) system to confirm that the produced protein is the target protein. According to the analysis, it was concluded that the purified protein was the target DNA polymerase. In order to determine if the purified protein is correctly folded or not, the secondary structure analysis of the protein with a purity over 90% was performed using Circular Dichroism (CD) in the far-UV (<260 nm) range. As a result of the study, the protein showed an apparent α-helix secondary structure with two minima at 208 and 222 nm wavelengths and a maximum at 190 nm wavelengths. Functional analysis of the protein on its folded state was completed by performing the Polymerase Chain Reaction (PCR). The 825 bp DNA region with 49.6% G-C content, and 1947 bp DNA region with 61% G-C content was amplified. These regions were selected considering the processivity of the enzyme. No-template control was used as negative control. The amplified regions were analyzed comparatively with commercial enzymes and it was observed that the target regions were successfully amplified. Commercially available high-fidelity DNA polymerase enzymes do not have endonuclease contamination and exonuclease contamination. Within the scope of quality control experiments, both endonuclease and exonuclease contamination of three biological replicates of our high-fidelity DNA polymerases were compared with commertial ones. λ DNA and λ DNA digested with HindIII were used as positive control. As a result, it has been shown that commercial enzymes and our high-fidelity DNA polymerase enzyme do not have endonuclease and exonuclease contamination. Another important test for demonstrating the quality of commercial enzymes is testing whether the protein remains stable under different conditions. For testing the stability of the polymerase, the effect of freeze-thaw stress repeated 20 times and also the effect of incubation of the enzyme for 5 days at room temperature were evaluated. As a result of these experiments, it has been shown that the freeze-thaw process during the general use of purified enzyme does not cause a negative effect on the activity of the protein, and that if the purified enzyme is forgotten at room temperature for up to 5 days during use, they can polymerize without reducing their activity. In conclusion, with this study, we have produced high-fidelity DNA polymerase with the same stability and processivity as commercial polymerases produced by large biotechnology companies with high efficiency and purity.
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ÖgeChronological lifespan analysis of stress-resistant yeasts(Graduate School, 2024-06-28)Saccharomyces cerevisiae has an important place in human life with its wide usage in various processes like fermentation, brewing, bread and wine production from the oldest times in history. Since its genome sequencing in 1996, it has become one of the most well-known and studied model organisms in many different areas of biology such as cell biology, biotechnology, cancer and aging research. Compared to other model organisms, its ease in genetic manipulation and cultivation conditions made it a convenient host for the production of heterologous proteins and economically valuable products. Yeast shares 30% of homology with many human genes, thus it is a convenient platform to study eukaryotic cell metabolism along with disease models. Aging is a common process all living organisms share and involves numerous metabolic and physiological changes that usually result from accumulated damage and deterioration. Despite the developed technology and improved living conditions through every passage of human life, it is estimated that the aging population will cover one-fifth of the whole population of the world by end of the century and age related diseases will cause a socioeconomic burden to governments. Studying aging in humans is complicated because of the long lifespan and economic and ethical concerns. Although a wide range of organisms share similar aging patterns, using yeast provides a convenient and accurate eukaryotic model with a shorter lifespan and easier growth capability. There are two main approaches to studying aging in yeast: chronological lifespan (CLS) and replicative lifespan (RLS). CLS seeks to analyze the lifespan of undivided yeast cells after they enter the stationary phase, mostly caused by decreased nutrients or toxic metabolite accumulation. RLS defines the number of cell divisions a cell undergoes before its death. CLS analysis is particularly useful for analyzing the response and survival of the cell against certain stress factors and modeling G0 cells that arrest their cell cycle. Metabolic engineering is an effective biotechnological approach for improving metabolic processes and product formation of the organism by altering the existing mechanisms or introducing new ones through the usage of recombinant DNA technology. In classical metabolic engineering, information on metabolic, genetic and physiological data of the strain is gathered, then the manipulations on relevant factors are employed to obtain the desired phenotype. Yet in the inverse metabolic engineering approach, for example when evolutionary engineering is employed, the desired phenotype is achieved using laboratory-based evolutionary settings. In this strategy, yeast strains can become resistant to certain stress factors or produce desired molecules throughout the increased stress treatment during culture. After obtaining the desired phenotype, yeast strains are examined by genomic or transcriptomic analyses to further determine the molecular changes in the genome or transcriptome. With more advanced xix technologies such as CRISPR-Cas9, altered genetic traits can be transferred to wild type strains to evoke the same resistant phenotype. In this study, previously obtained stress-resistant S. cerevisiae strains were analyzed for their CLS and viability performances. Stress factors selected for this purpose were antimycin, boron and freeze-thaw stresses which can affect the production efficiency or viability of yeast strains. In parallel with the general evolutionary engineering strategy, strains were obtained by increasing the stress levels gradually, which is the concentration of the compound in the case of antimycin and boron, and repeat numbers in the case of freeze-thaw stress, in selection cultures until the resistant population is achieved. It was shown previously that the evolved strains could become cross resistant to other stress types or their longevity can be affected by the process. The aim of this study was to determine which type of stress resistances can extend or shorten the CLS of the yeast thus affecting the lifespan of the industrial and laboratory yeast strains. For this aim, both quantitative and semi-quantitative CLS analyses were carried out. In the semi-quantitative CLS experiment, OD600 values of the yeast strains were set to 6 before they were spotted onto agar plates every 2nd day with serial dilutions. The longevity of the resistant strains was compared with their control strains visually, based on the growth on the plates. According to the results, P8 which is the freeze-thaw stress-resistant, industrial polyploid strain had a longer CLS than its industrial reference strain, whereas the antimycin and boron-resistant strains did not have a longer CLS than their reference strain. In the second part of the study, quantitative CLS analysis was done by spreading the long-lived industrial P8 strain along with its industrial reference strain R625 and the laboratory reference strain 905 to agar plates. The longevity was measured by counting colony-forming units (CFUs). The experiment was repeated until the viability of the cultures was reduced to 0.0001% from day 0 of the experiment where the viability was accepted as 100%. In the second part of the study, further validating the results from the semi-quantitative analysis, P8 exhibited longer CLS compared to its industrial reference strain and could live until the 10th day of the experiment. Among the various stress-resistant strains tested in this study, only the freeze-thaw stress-resistant, industrial P8 strain was found to have a longer CLS. However, the antimycin and boron-resistant yeast strains did not show a longer CLS compared to their laboratory reference strain. Since the freeze-thaw response was previously associated with oxidative stress response and nutrient metabolism alterations, the longer CLS of the freeze-thaw stress-resistant industrial strain can be related to changes in respective pathways that originated from the evolutionary engineering process. In the scope of the research done for this study, despite being studied in other organisms, the effect of boron resistance on longevity was studied in yeast for the first time. Similarly, antimycin resistance was examined for its effect on longevity in yeast for the first time, as well. Further studies to analyze genomic and transcriptomic changes that occurred by the acquired resistance can be performed and these changes can be transferred to wild-type or reference strains to assess the viability and CLS profiles.
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ÖgeComparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain(Graduate School, 2022)Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.