Theses supervised by Prof. Dr. Zeynep Petek Çakar

Rızıq Nahıdh Ismaeel Alsamawı

Investigation of antimicrobial resistance in wild-type and stress-resistant Saccharomyces cerevisiae strains

Fungi are organisms that live in various habitats like air, soil and water. Some species live as symbionts and some are parasites. Fungi kingdom consists of mushrooms, molds and yeasts. They all contain rigid cell walls. Yeast are spread worldwide, they are mostly in the phylum ascomycota, and only a few are within the phylum Basidiomycota. There is a high number of economically important types of ascomycete yeasts. These types are commonly used in bread, wine, and beer production; and are selected from Saccharomyces cerevisiae strains. Saccharomyces cerevisiae is a eukaryotic unicellular microorganism. It has a completely sequenced compact genome which makes it a suitable microorganism for easy manipulation of the genome and also ease of cultivation. In this study, ethanol-resistant, coniferyl aldehyde-resistant, caffeine-resistant, propolis-resistant and antimycin-resistant mutant S. cerevisiae strains which were previously obtained by inverse metabolic engineering strategy, were analyzed against different antimicrobials and possible relations between resistance to those antimicrobials were investigated. For this analysis, spot assay method was applied to reference strain (905) and mutant strains under respirative and fermentative conditions against rapamycin, erythromycin, kanamycin and antimycin-A antimicrobials. Antibiotics are generally known and referred to as a subset of anti-infective agents which are derived from bacterial or fungal sources that are used to treat bacterial infections. Antibiotics are generally used medically to treat or prevent bacterial infections. Most antibiotics inhibit the replication or completely kill bacteria. The name 'antimicrobial' is a general term for any compound that inhibits some vital functions or kills microorganisms including chemicals such as antibiotics, antifungal agents, antivirals, and antiseptics. Fungi and viruses may also pose some risks to humans and are targeted by antifungals and antivirals, respectively. Rapamycin is a macrocyclic antibiotic. It is produced by the soil bacterium Streptomyces hygroscopicus found on Easter Island soil. Rapamycin was discovered as a potent antifungal agent, but initially, it exhibited an undesirable and immunosuppressive effect, leading to its development as a clinically useful drug. Rapamycin is the inhibitor of Target of Rapamycin (TOR) pathway. This pathway has important roles such as nutrient sensing, chronological aging, apoptosis and energy metabolism. There is a recent discovery which states that the mTOR inhibitor rapamycin can extend the lifespan of mammals which has created great excitement, as it represents the first demonstration of the pharmacological extension of maximum life in a mammalian species. Since then, rapamycin effects on the lifespan of mammals have been confirmed by some additional studies. Kanamycin is mostly known and referred as kanamycin (A). It is an aminoglycoside bactericidal antibiotic which is found in oral, intravenous, and intramuscular forms. It is used to treat and cure a wide range of infections. Kanamycin is isolated from the bacterium Streptomyces kanamyceticus, and its mostly and commonly used form is kanamycin sulfate. Aminoglycosides function by binding to the bacterial 30S ribosomal subunit and causing misreading of t-RNA. By this way, the bacterium cannot synthesize the proteins vital for bacterial growth. Aminoglycosides are generally used against infections of aerobic, gram-negative bacteria such as Pseudomonas, Acinetobacter, and Enterobacter. Additionally, some mycobacteria are susceptible to aminoglycosides, including bacteria that cause tuberculosis. Infections caused by gram-positive bacteria can also be treated with aminoglycosides, but other antibiotics are more potent and less harmful to the host. Erythromycin is a bacteriostatic macrolide antibiotic produced by Streptomyces erythreus. By binding to the 50S ribosomal subunit, it inhibits protein synthesis. The binding to the 50S ribosomal subunit inhibits peptidyl transferase activity and interferes with the translocation of amino acids during the cycling and coupling of proteins. Erythromycin is released from the bacterial cell membrane and is reversibly attached to the 50S subunit of the bacterial ribosome. This prevents bacterial protein synthesis. Depending on the concentration of the drug, erythromycin may exhibit bacteriostatic or bactericidal action in the infection site and the sensitivity of the related organism. Additionally, erythromycin may enhance the actions of other drugs by the inhibition of microsomal metabolism, leading to toxicity of these other medications. Erythromycin is available in the form of different esters, including erythromycin estolate, erythromycin lactobionate, erythromycin ethylsuccinate, and erythromycin gluceptate. Antimycin-A is an antifungal produced by Streptomyces species. It is the mitochondrial complex III inhibitor. Antimycin binds closely to a pocket in 1 of 5 of the major electron transport proteins. Antimycin binds at the stage where ubiquinol, referred to as (coenzyme Q,) which usually binds to produce electrons to the level of (O2) that is bound to a close iron-containing enzyme. This explains why the electron transporter is blocked at some stages, the bound oxygen is exchanged to superoxide, a very reactive form of oxygen. Antimycin interacts specifically with the complex system of protein structures related to the electron movement. Biochemists make use of this specific binding of antimycin to avert the electron flow and to investigate the chemical details of aerobic respiration. With these investigations, significant information has been revealed about how antimycin adds at the enzyme level, down to the molecular level and the specifics on how the side chains on the antimycin can impact and influence the electron movement at the protein stage. Antimycin is an active ingredient in Fintrol®, a registered product, used as an industrial agent (pesticide). As a result of the antifungal properties, it has been demanded for commercial applications in agriculture. Antimycin is highly toxic to fish species. However, it is used in many fisheries. It has also been used as a commercial toxic agent to get rid of the unwanted fish species in catfish farms. Cross resistance analysis revealed that all mutant strains showed similar behavior against erythromycin and antimycin-A stress. Additionally, under fermentative conditions, only rapamycin affects the growth of the yeast. The aim of this study was to determine the antimicrobial resistances of some stress-resistant S. cerevisiae mutant strains and to identify possible relationships between cross-resistances to different antimicrobials and a variety of stress types. The results could be useful for future research on antibiotics and antifungals resistance.

İstanbul Technical University · Institute of Graduate Studies in Science

2018

Lactobacillus plantarumLactic starter

Evolutionary engineering of polyphenol resistance in lactic acid bacteria

The aim of this study was to obtain robust starter cultures for more efficient olive fermentations by developing stress-resistant lactic acid bacteria, more preferably Lactobacillus plantarum strains by using evolutionary engineering approach. Since resistance mechanisms in lactic acid bacteria involve numerous genes and many regulators, it would be difficult to obtain the desired resistant phenotypes by direct genetic engineering or over-expressing certain genes. Thus, evolutionary engineering could be a good alternative strategy to obtain the desired phenotypes. In this approach, the resulting strains are not considered as genetically modified organisms which makes them more acceptable for the food industry. It was generally thought that lactic acid bacterial starter cultures for olive fermentations were not robust enough due to the presence of olive polyphenols and mainly oleuropein in fermentation brines. In the first part of this thesis, this hypothesis was tested by determining the resistance of various strains to stress factors that are commonly encountered during olive fermentations. The results revealed that NaCl, and not oleuropein, was mainly responsible for the decreased growth rates of lactic acid bacteria during olive fermentations. For the evolutionary engineering studies, a L. plantarum strain (T129) isolated from table olive-producing companies in the Marmara Region was obtained from TUBITAK MAM Food Institute collection and a L. plantarum type strain DSMZ 10492 isolated from olive-processing brines in Portugal was obtained from DSMZ Culture Collection. These strains were treated with EMS (ethyl methane sulfonate) to obtain two different, genetically diverse initial populations for selection. The NaCl resistance of these populations was increased by applying increasing levels of NaCl stress during successive batch cultures of selection. Mutant individuals were selected from the final mutant populations that resisted 10 % (w/v) NaCl. The survival rates of mutant individuals were compared with those of the wild-type at 10% (w/v) NaCl and 5 g/l oleuropein-containing 10% (w/v) NaCl. Most of the mutant individuals survived both NaCl and oleuropein stress, unlike their wild-type strains. The cross-resistance of selected mutant individuals against commonly encountered stress factors during olive fermentations were determined to confirm the suitability of the mutants as robust olive starter cultures. One mutant strain derived from L. plantarum DSMZ 10492 (D2) and one mutant strain derived from the L. plantarum T129 (T8) were chosen for further studies. The NaCl resistance of the mutant strains during five successive cultivations in non-selective (stress-free) media were determined and the results confirmed that NaCl resistance was not an adaptation, but had a heritable genetic background. These genetically stable strains could therefore be used as starter cultures for olive fermentations without losing their gained resistance. Another aim of this thesis study was to obtain lactic acid bacteria that could be used as probiotic cultures for olive fermentation. Such a starter culture would not only ensure a standardized olive fermentation, but also provide added health benefits to its user. Thus, lactic acid bacteria need to be engineered to increase their survival during the passage through the upper digestive tract. It is difficult, however, to select a single stress condition to obtain efficient probiotic mutants. Ingested microorganisms are faced with a series of stress factors during their passage through the gastrointestinal tract. Time-based decrease in pH and the presence of gastric enzymes are major stress factors in the stomach. While peristaltic movement of the small intestine prevents microbial colonization, weakened bacteria are faced with pancreatic enzymes, bile salts and nutrient deprivation in different sections of the small intestine. Thus making use of an in vitro model of the upper gastrointestinal tract was the novel evolutionary engineering strategy to select for more viable, robust and efficient probiotic strains. It was hypothesised that mutant individuals with high survival rates would be enriched during the consequent passages through the in vitro model. By this strategy, it would be possible to isolate an individual with increased survival. The TNO in vitro model of the stomach and small intestine (TIM-1) is a computer-controlled dynamic model that accurately mimics the dynamic conditions in the upper parts of the human digestive system. TIM-1 consists of four compartments: the stomach, duodenum, jejunum and ileum. It mimics body temperature, peristalsis, intestinal passage, specific pH conditions, enzyme and electrolyte levels as close as possible to the physiological conditions. Therefore, the EMS-mutagenized rifampicin-resistant mutant population of T129 was introduced to this system for five successive times. At the end, an individual strain, J1, was selected with a survival rate close to 100%. The genetic stability of this strain was determined by reintroducing this strain to the TIM-1 system after stress-free cultivation. The survival in the upper gastrointestinal tract was preserved which showed that the mutant strain was genetically stable. Finally the genetic background of the increased survival of the J1 strain was investigated by comparing the whole genome sequences of the wild type T129 and The mutant strain J1. It was confirmed that all mutations were either G to A or C to T, as expected typically for EMS mutagenesis. The whole genome was annotated and the genes that were affected by these mutations were determined as: phosphoribosylformylglycinamidine synthase subunit PurL, glutamine ABC transporter, permease protein, DNA repair protein RecN, ribosome small subunitdependent GTPase A, molecular chaperone DnaK, DNA-directed DNA polymerase III, alpha chain PolC-type, phosphatidate cytidylyltransferase cell surface protein, Cardiolipin synthetase and ATP-dependent zinc metalloprotease FtsH. This revealed that changes in cellular metabolism, DNA repair, and cell wall composition might possibly have led to the increased survival of the mutant strain J1. To conclude, novel selection strategies were successfully applied in this study to obtain stress-resistant and robust L. plantarum strains with improved probiotic properties for potential use in olive fermentations. Further transcriptomic and proteomic research would be necessary to understand the complex molecular basis of the desirable phenotypes.

Tarık Öztürk

İstanbul Technical University · Institute of Graduate Studies in Science

2018

Bakers yeastMicroarray analysisMicroorganisms

Molecular and physiological characterization of iron resistant Saccharomyces cerevisiae

Iron, the fourth most abundant element in earth crust, is essential for living organisms. At high concentrations, however, it is toxic. The electron transport potential between its two most common oxidation states (Fe3+ and Fe2+) makes iron crucial for all living organisms. Iron ions play critical roles in oxidative phosphorylation. Iron shows similar physical and chemical properties with other transition metals, like copper, zinc, nickel and cobalt. Transition metal ions have common transport systems or they can replace each other in some structures. Thus, it can be expected that iron resistance and resistance to other transition metals may have common features. The aim of this study was the molecular and physiological characterization of an iron-resistant Saccharomyces cerevisiae mutant that was previously obtained by evolutionary engineering, an inverse metabolic engineering strategy. For this purpose, the cross-resistance of the iron-resistant strain against other stress types (particularly other transition metal ions) were determined in detail. Growth physiological analyses were also performed, which revealed that the iron-resistant mutant produced high levels of glycerol and accumulated high amounts of trehalose. The yeast S. cerevisiae can survive toxic metals by either of the two major kinds of resistance mechanisms: sequestration or avoidance. To gain insight into the major iron resistance mechanism in the resistant strain, iron contents of the mutant and the reference strain were determined both in the presence and absence of iron stress, by using Flame Atomic Absorption Spectrophotometry (F-AAS). The results showed that the iron-resistant yeast strain had significantly higher iron level than the reference strain, already in the absence of any iron stress. When iron stress was present in the culture medium, however, the iron-resistant mutant did not take up as much iron ions as the reference strain did. These results imply that in the presence of iron stress, the iron-resistant mutant avoids iron ion uptake, to survive iron stress conditions. For molecular characterization of the iron-resistant S. cerevisiae strain, transcriptomic analyses were performed using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and microarray technology, both in the presence and absence of iron stress. Microarray analysis results showed that the iron-resistant S. cerevisiae strain had higher expression levels of the oxidative stress-related genes than the reference strain, even in the absence of any iron stress. According to the results obtained by the microarray analysis, lipid peroxidation assay and intracellular reactive oxygen species (ROS) levels determination assay were performed. The intracellular ROS level of the iron-resistant strain was found to be lower than that of the reference strain. Thus, the iron-resistant strain possibly expresses high levels of oxidative stress-related genes and produces antioxidant c which reduce intracellular ROS levels. By this means, it may survive high levels of iron stress.

Berrak Gülçin Balaban

İstanbul Technical University · Institute of Graduate Studies in Science

2016

İstanbul Technical University · Institute of Graduate Studies in Science

Proteomic and genetic analyses of heat-resistant Rhodobacter capsulatus mutants with modified hydrogen production capacity

Hydrogen is a clean energy carrier whose importance in energy systems has been increasing, especially for the last two decades. The biological hydrogen production by using photoheterotrophic microorganisms is one of the cleanest methods of hydrogen production. To overcome efficiency losses due to overheating in outdoor photobioreactors, heat-resistant microorganisms are desirable. In this study, a combination of the comparative whole genome sequencing, comparative proteomic, and transcriptomic analyses of two previously obtained Rhodobacter capsulatus heat-resistant mutants; A52 and B41, with decreased and elevated hydrogen production capacities, respectively, have been performed to identify mutations and expression differences related to desired higher hydrogen production capacity and heat resistance. Uncovering the molecular mechanisms of resistance to high temperature besides hydrogen production by R.capsulatus will be a reference for the development of strains resistant to heat stress. Also the characterization of mechanisms affecting the production of hydrogen in R.capsulatus allowed the determination of target genes and metabolic pathways for the creation of high-efficiency hydrogen producing mutants through metabolic engineering. Genomic analyses showed a small group of mutations which was directly related to hydrogen production metabolism. In the mutant B41 genome, mutations were characterized in nifD, nifJ, glnD, nifB1, ccpA, hupD, dmsA and cbbR1 genes, on the other hand; in the A52 mutant, mutations were characterized on the nifB2, rnfF, nifJ, cbbO, anfH, amt, moeA, and hupD genes. Proteomic analyses revealed possible regulatory functions of the hypothetical proteins RCAP_rcc03215, RCAP_rcc03277, and RCAP_rcc00730 in hydrogen production metabolism of R.capsulatus. The effects of nitrogen metabolism-related mutations were tested by quantitative real-time polymerase chain reaction (qRT-PCR) on the expression levels of nifA gene (the activator of other nitrogen fixation genes), nitrogenase structural gene nifH, nitrogenase regulator ntrC gene, RubisCO structural genes cbbL, cbbS and cbbO, nitrogenase iron protein encoding gene anfH, hydrogenase maturation protease encoding hupD, molybdopterin biosynthesis protein encoding moeA, ammonium transporter gene amt, and dimethyl sulfoxide reductase, A subunit encoding dmsA genes. The most promising mutation was the one found on the glnD gene of B41 strain which is the highest hydrogen-producing strain among the heat-resistant mutants. This gene encodes PII uridylyltransferase and the effect of this mutation was confirmed by quantitative polymerase chain reaction (qRT-PCR) analyses.

Abdulmecit Gökçe

2017

İstanbul Technical University · Institute of Graduate Studies in Science

Molecular and physiological investigation of longevity in yeast

World population has changed in both developed and developing countries due to low birth and death ratio, resulting in relatively elder populations, which is a major challenge. Aging is a risk factor for several diseases such as Alzheimer, Parkinson, diabetes, cancer and cardiovascular diseases, due to declining cellular physiological functions with time. In recent years, researchers have been focusing on this issue due to the increasing socioeconomic concerns. The purpose is to understand the cellular aging process and to provide healthy lifespan. In aging research, the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, the worm Caenorhabditis elegans, rodents and primates have been used as model organisms. The yeast S. cerevisiae is a unicellular eukaryotic microorganism and it has been used in industrial processes such as baking, wine and beer production since ancient times. It has also been commonly used as a eukaryotic model organism in genetic and molecular biological research since it does not need expensive and special media and cultivation conditions. The yeast S. cerevisiae is a commonly preferred model organism in aging studies because of the shortness of its life span, availability of its genome sequence data and extensive molecular tools and techniques, and its similarity to mammalian cells. Aging studies can be performed by two ways in S. cerevisiae: by investigating the replicative life span (RLS) or the chronological lifespan (CLS). RLS has been developed because of the asymmetrical division of S. cerevisiae cells. In the method, after mitosis, the daughter cell is separated from the mother cell by using a micromanipulator, and the procedure is continued until the mother cell stops division. At the end of mitosis, the number of buds one mother cell produced are determined and the number of daughter cells is the RLS of S. cerevisiae. CLS is the length of time where non-dividing stationary phase yeast cells survive. In the method, cell viability at the beginning of the stationary phase of growth is determined by viable cell counting method on solid media. After that, viability of non-dividing cells at the stationary phase is monitored. Since (i) stationary phase cells produce energy from mitochondria, (ii) damage accumulates over time and (iii) cells exit from the cell cycle (G0), CLS method is accepted as a convenient model to study aging of non-dividing cells (e.g., neurons) in higher eukaryotic organisms. Long-lived S. cerevisiae, C. elegans and Drosophila mutants have had a major role for understanding cellular aging mechanism. The studies share a similar approach, since the long-lived mutant strain is obtained first, and then it is investigated in detail to understand its molecular basis. These strategies show similarity to inverse metabolic engineering approach. In inverse metabolic engineering approach, first, the desired phenotype is obtained, and then, its genetic mechanism is identified. This approach is a very powerful strategy to obtain complex desired microbial phenotypes with genetic complexity. So far, the approach has not been used to obtain chronologically long-lived yeast. In the present study, inverse metabolic engineering approach was used to obtain chronologically long-lived S. cerevisiae. Furthermore, since previous aging research with model organisms has shown a close relationship between stress resistance and longevity, in the current study, CLS of previously obtained stress-resistant S. cerevisiae mutants was also investigated. To isolate a chronologically long-lived yeast mutant by using inverse metabolic engineering, first, genetic diversity of the initial population for selection was increased by ethyl methane sulfonate (EMS)-mutagenesis. Successive batch cultivation of the initial mutant population was performed under gradually enhanced caloric restriction, since caloric restriction is known to extend lifespan. During successive batch cultivations, glucose concentration of the medium was decreased from 0.5% to 0.02%. In the last batch cultivation, since growth ratio of the mutant population decreased to 10%, the selection procedure was ended. After that, individual mutants were randomly picked from the last population. Semi-quantitative and quantitative CLS analyses were applied to these individual mutants. A single mutant (called SRM11) that has the highest chronological survival among the other mutants was chosen for further physiological and molecular analysis. Quantitative CLS method based on viable cell counting on solid media is tedious for large studies where multiple strains and multiple conditions are tested, as a high number of plates and flasks is required for incubation. It is also labor-intensive. Therefore, in the present study, the semi-quantitative method was used for preliminary determination of CLS in mutants. A culture density adjustment step was introduced to the semi-quantitative method, for a more reliable evaluation of CLS of cultures at significantly different culture densities. Following the semi-quantitative CLS, quantitative CLS analysis was applied to the prominent mutants. The procedure was applied successfully and correctly determined the chronologically long-lived phenotypes in a wide range of strains and conditions. Growth behavior of the mutant SRM11 was investigated by growth curve and biomass analysis. The purpose of these experiments was to determine whether any trade-off in growth physiology occurred or not. Analysis results showed that there was no trade-off in the growth fitness of the mutant SRM11 in both standard medium (2% YMM) and calorie-restricted medium (0.5% YMM). In this experiment, it was observed that 75% reduction of glucose concentration of the medium (from 2% to 0.5% glucose) did not affect growth ratio of the yeast strains when glucose was utilized. High-Pressure Liquid Chromatography (HPLC) analysis was performed to investigate the metabolic profiles of the long-lived mutant SRM11 and the reference strain. For this purpose, glucose, ethanol, glycerol and acetate concentrations in culture samples were monitored by HPLC analysis. Even though there was no significant difference in glucose consumption between the reference strain and the mutant SRM11, there was a significant difference in the production of fermentative metabolites. The mutant SRM11 produced low amounts of ethanol, glycerol and acetate in standard glucose medium (2% YMM). These findings indicated a respirative shift in the metabolism of the mutant SRM11, showing that respirative metabolism is related to chronological longevity in S. cerevisiae. In 0.5% YMM, a limited ethanol and glycerol production was observed in the cultures of the reference strain and the mutant SRM11, which indicates that at 0.5% glucose concentration, fermentation is limited. Aging studies have shown a close relationship between stress resistance and longevity in model organisms. Therefore, stress resistance analysis of the long-lived mutant SRM11 was performed, using the semi-quantitative spot assay method. The results revealed that the mutant SRM11 highly resisted to copper stress. Also, SRM11 resisted to silver, phenylethanol, ethanol and boric acid stresses, indicating that these stress types may be related with chronological longevity. Whole genome transcriptome of the long-lived mutant SRM11 was analyzed by using DNA microarray technology, to understand the molecular mechanisms of chronological longevity in yeast. DNA microarray analysis results revealed that 769 open reading frames (ORFs) were upregulated, 838 ORFs were downregulated by two-fold and higher in the long-lived SRM11 mutant, compared to the reference strain. For the interpretation of the gene expression change, Gene Ontology (GO) analysis was used. GO analysis of the upregulated genes indicated that oligosaccharide-carbohydrate metabolic biological process and response to oxidative stress were induced in SRM11. Furthermore, GO analysis of the downregulated genes revealed that ribosome subunits biogenesis, ribosome assembly and RNA processing were repressed in SRM11. These results suggest that repression of protein synthesis and activation of oligosaccharide-carbohydrate metabolic processes and response to oxidative stress are associated with the extension of chronological longevity in the yeast S. cerevisiae. CLS of various stress-resistant S. cerevisiae mutants was also investigated by semi-quantitative and quantitative methods. Among the tested stress-resistant mutants, silver-resistant, ethanol-resistant, phenylethanol-resistant, oxidative stress-resistant mutants were determined as long-lived mutants. Resistance to oxidative stress has been shown to be related with chronological survival in yeast, however; resistance to silver, ethanol and phenylethanol stress has not been shown, yet. The present study, for the first time, showed that resistance to silver, ethanol and phenylethanol may be related with chronological longevity in the yeast S. cerevisiae. In the current study, a comparative transcriptomic analysis of the long-lived stress resistant mutants (silver-resistant, phenylethanol-resistant and ethanol-resistant mutant and long-lived mutant SRM11) was also carried out to find a relationship between chronological longevity and stress resistance. The comparative transcriptomic analysis showed that 17 downregulated genes and 48 upregulated genes were overlapping in the long-lived and stress-resistant mutants. GO analysis of the overlapping upregulated genes showed that the upregulated genes were enriched in mostly oligosaccharide and carbohydrate metabolic process and generation of precursor metabolites and energy. GO analysis of the overlapping downregulated genes indicated that ribosomal subunits biogenesis and aminoacid transport biological process were repressed. As a conclusion, the present study suggests that increased respirative metabolism, resistance to copper, silver, ethanol and phenylethanol stress, repression of protein synthesis and induction of oligosaccharide and carbohydrate metabolic processes are associated with chronological longevity in the yeast S. cerevisiae. Further detailed analyses at genomic and proteomic levels will help understand the complex molecular basis of the longevity in S. cerevisiae.

Saccharomyces cerevisiae

Mevlüt Arslan

2017

Gene cloningGenetic vectorsYeast killer+2

Kpkt katil toksininin Saccharomyces cerevisiae ve Pichia pastoris hücrelerinde heterolog üretimi için ekspresyon vektörlerinin oluşturulması

In nature, microorganisms process several defense mechanisms to survive under limited nutritional conditions. Killer yeasts have one of the defense mechanisms. They produce toxins called killer toxins (KTs) against sensitive microorganisms. Killer toxins are produced by killer yeasts into extracellular environment and they show their activities by binding to specific compounds called killer toxin receptors (KTRs). Killer toxins can be produced as either proteins or glycoproteins. While killer yeasts have self-immune system to their own KTs, their cytocidal activities usually process in a two-step mechanism on sensitive strains. In the first step, they bind the receptors on cell-wall of the sensitive strains. These receptors are known as primary KTRs. Secondary KTRs found on cell membrane are the second targets of KTs. They bind to these receptors to kill the sensitive strains to enter the cell. Different action mechanisms have been presented for killer toxins and they depend on the genetic determinants as well as physical-chemical properties of KTs. Kpkt is one of the killer toxins produced by Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii). Kpkt killer toxin is active against wine spoilage yeasts such as Kloeckera apiculata and Hanseniaspora uvarum. Therefore, Kpkt has a potential to be used in wine fermentation since it maintains its zymocidal activity for more than 14 days in wine. Kpkt is a glycoprotein and it has a β-glucanase activity. It shows its activity by hydrolyzing β-1,3- and β-1,6- glucans on cell-walls of the sensitive strains. Kpkt killer toxin is encoded by TpBGL2 gene in T. phaffii. The aim of the study was to construct expression vectors for heterologous production in Saccharomyces cerevisiae and Pichia pastoris. For this purpose, pYES2.1/V5-His-TOPO® vector was utilized for both extracellular and intracellular production in S. cerevisiae as well as pPIC9 vector was utilized for extracellular production in P. pastoris. In the first part of the study, suitable yeast strains resistant to Kpkt were selected for heterologous production of Kpkt. S. cerevisiae YPH501 and BY4741 along with P. pastoris GS115 strains were subjected to killer plate assay using YPD plates containing citrate-phosphate buffer (pH 4.6). P. pastoris GS115 appeared as a suitable strain after this plate assay. Then, S. cerevisiae YPH501 and BY4741 were grown in galactose containing media to observe their growth profiles in galactose containing media since pYES2.1/V5-His-TOPO® vector harbors GAL1 promoter which is active only in the presence of galactose. Finally, BY4741 was selected as a suitable strain for transformation. In this study, three different vectors were constructed: i) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter; ii) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the  sequence; iii) TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the  sequence. To construct them, first DNA extraction was carried out from T. phaffii. Next, DNA quality was checked by PCR amplifying with ITS1 and ITS4 universal primers. First, TpBGL2 gene was amplified using BGL2F and BGL2R primers from T. phaffii total DNA in order to ligate it directly to pYES2.1/V5-His-TOPO® vector to construct the first vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter) for intracellular expression in S. cerevisiae BY4741. After ligation, vectors were transformed into Escherichia coli cells and vectors were extracted from E. coli cells through minipreparation. The resulting vectors were further analyzed to investigate both the presence and the orientation of TpBGL2 gene in the vectors by constructing restriction patterns with MlyI and XbaI enzymes as well as by performing sequence analysis. In addition, PCR amplification was carried out using GAL1F and BGL2R primers. All results confirmed that the insert was in frame with GAL1 promoter. To construct the second and the third vectors, first TpBGL2 was amplified from T. phaffii total DNA with FW2 and RV2 primers having EcoRI and NotI restriction sites, respectively. TpBGL2 gene flanked by EcoRI and NotI restriction sites was ligated into pGEM-T Easy Vector by TA cloning. After transformation and cloning in E. coli cells, extracted primers were digested by EcoRI to confirm the presence of the insert. Next, both empty pPIC9 vectors and pGEM-T Easy vectors having TpBGL2 gene flanked by EcoRI and NotI restriction sites were digested by EcoRI and NotI enzymes. Then, they were ligated to construct the third vector (TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the  sequence). Both sequence analysis and PCR amplification with AOX1-BGL2R primer set confirmed that the insert was in frame with AOX1 promoter and  sequence. To construct the second vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the  sequence), TpBGL2 gene downstream the  sequence was amplified using ALFA and BGL2R primers and the amplicon was ligated into pYES2.1/V5-His-TOPO® vector. However, sequence analysis showed that the insert was in wrong orientation and no further studies were carried out with this vector. pYES2.1/V5-His-TOPO® + TpBGL2 vector was transformed into S. cerevisiae BY4741 strain for intracellular production of Kpkt killer toxin and the presence of the insert was confirmed by PCR analysis in S. cerevisiae transformants. The selection was carried out on YNB selective medium without uracil plates. One of the six transformants called T1 was cultivated in 2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium. After 24 h and 48 h, sampling was carried out for crude extraction and for taking supernatant from the culture. Next, well plate assay was performed to investigate killer activity on buffered YPD plates spread by sensitive S. cerevisiae DBVPG 6500 strain to Kpkt killer toxin. T1 was also cultivated in 0.2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium for 48 h and it was transferred into 2% (w/v) galactose containing minimal medium. After 24 h incubation, well plate assay was performed again for both crude extract and the supernatant of the culture. In addition, T1 was cultivated in 2% sucrose (w/v) containing buffered selective minimal medium and it was transferred into 2% (w/v) galactose containing buffered selective medium after 48 h of growth. Well plate assay was performed again after 24 h of growth in galactose containing medium. All studies showed that Kpkt killer toxin did not perform its killing activity when it was expressed intracellularly. Finally, the third vector (PIC9 + TpBGL2) was transformed into P. pastoris GS115 cells for extracellular expression of Kpkt in P. pastoris. To do that, P. pastoris cells were prepared to obtain competent cells for transformation which was carried out through electroporation. For transformation, linearized plasmids obtained by digesting with either SacI or BglII enzyme were utilized along with linearized empty pPIC9 vectors with the same enzymes. Selection was carried on minimal dextrose plates. Next, the transformants were grown on both minimal dextrose and minimal methanol plates to determine putative Mut+ (Methanol utilization plus) or MutS (Methanol utilization slow) phenotypes of the transformants since only Mut+ phenotypes can grow on methanol containing medium if the insertion results in functional AOX1 gene encoding alcohol oxidase, which is the main enzyme in methanol utilization pathway. Finally, transformants were subjected to PCR amplification using AOX1 and AOXR primers to investigate whether they have inserts. The evaluation of the killer activity of Kpkt killer toxin in P. pastoris is in progress.

Murat Üstün

İstanbul Technical University · Institute of Graduate Studies in Science

İstanbul Technical University · Fen Bilimleri Enstitüsü

Evrimsel mühendislik yöntemi ile elde edilen kobalta dirençli Rhodobacter sphaeroides bakterisinin fizyolojik analizi

Rhodobacter sphaeroides is a gram negative purple non-sulfur α-proteobacterium found in soil, mud, deep lakes and stagnate waters. It has metabolic diversity, as it is able to grow under photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic conditions. It can alter its metabolism according to the changing environmental conditions and nutrient deficiency. Due to its metabolic versality, sequenced genome and no requirement of unusual conditions for growth, it is commonly used in biotechnological applications. R. sphaeroides is capable of synthesizing and storing biodegradable and biocompatibile polyester polyhydroxybutyrate (PHB) as insoluble inclusion bodies in cytoplasm. Some of the other byproducts of R.sphaeroides in industry are 5-aminolevulinic acid, Coenzyme Q10, carotenoids, vitamin B12, indole terpenoids and plant hormones. Large scale H2 production in a photobioreactor, ZnS nanoparticle production for medical purposes and potential usage in bioremediation are also reasons for biotechnological importance of R. sphaeroides. R. sphaeroides has the ability to survive under changing stress conditions by modifying its cellular and genetic composition. For instance, oxidative stress causes down-regulation of the carotenoid genes, while heavy metal stress increases membrane integrity. In another study, cobalt-resistant R. sphaeroides population was obtained under gradually increasing cobalt stress conditions, using evolutionary engineering approach. The last (64th) population was able to survive 15 mM cobalt stress, with a survival rate of 21%. The last population was spread onto solid Luria Broth plate and eight colonies (called G1-G8) were selected from there. In this study, the selected individual mutants were phenotypically characterized.

Güneş Atay

İstanbul Technical University · Fen Bilimleri Enstitüsü

Donma-eri̇me stresi̇ne di̇rençli̇ Saccharomyces cerevisiae laboratuvar ve endüstri̇yel suşlarinin gen anlatim düzeyleri̇ndeki̇ farkliliklarinin qRT-PCR i̇le anali̇zi̇

The aim of this thesis study was to analyze gene expression level differences between freeze-tolerant laboratory and industrial strains of Saccharomyces cerevisiae, to gain insight into the molecular mechanisms of freeze tolerance in this yeast. For this purpose, S. cerevisiae laboratory strain '905', industrial strain 'R625' and their freeze-thaw resistant mutant individuals 'F1' and 'P8' were used. Freeze-thaw stress-related genes were chosen according to whole-genome transcriptomic analysis results of 905 and F1, which have been obtained previously. S.cerevisiae is a small, unicellular fungus that has been used as a model eukaryotic organism in biological research for a long time. Also, it has been commonly used in industrial processes since ancient times. The ability to ferment sugars makes the budding yeast is commercially important in bread-making and wine industries. The organism is also being used in bioethanol and fuel ethanol production, recently. S. cerevisiae is also named as baker's yeast because of its ability to leaven dough. It is also known that the yeast cells are exposed to freeze-thaw stress during long term storage conditions. Thus, cryopreservation process is a stressful condition for yeast cells. The freeze-thaw resistant mutants F1 and P8 were obtained in a previous study by an inverse metabolic engineering approach, based on random mutation and selection under freeze-thaw stress conditions. Whole-genome transcriptomic analyses of 905 and F1 were also done previously. To verify the transcriptomic analysis results obtained by DNA microarray technique, gene expression levels of selected genes were determined by Quantitative RT- PCR. The genes were selected from microarray data according to their high up- or downregulation levels. Highy upregulated genes HSP12, FMP45, HSP26, HXK1 and highly downregulated genes PHO84, NSR1, ZRT1 were chosen for gene expression analysis by qRT-PCR. The qRT-PCR experiments were performed under control and stress conditions for '905', 'F1' 'R625' and 'P8' strains. The expression profile of 'F1' was normalized to that of the wild type laboratory strain '905', and the expression profile of 'P8' was normalized to that of the wild type industrial strain 'R625'. In conclusion, it was found that freeze-thaw stress causes significant changes in gene expression patterns of both laboratory and industrial strains. In addition, some differences were also found between gene expression level results of microarray and qRT-PCR analyses. This indicates the importance of verifying DNA microarray results by qRT-PCR, as qRT-PCR is a more sensitive method.

Elif Engin

İstanbul Technical University · Institute of Graduate Studies in Science

Evrimsel mühendislik yöntemi ile Rhodobacter sphaeroides'in CoCl 2 stresine dirençli suşlarının eldesi

Rhodobacter sphaeroides is a metabolically diverse organism which is able to fix molecular nitrogen and produce H 2 as side product. It can grow under both oxic and anoxic conditions heterotrophically or photosynthetically. This wide repertoire of metabolism puts this organism in the centre of metabolic regulation and bioremediation studies. R. sphaeroides is used in the production of vitamin B 12 , coenzyme Q10, 5-aminolevulinic acid (ALA), porphyrin, as well as in bioremediation of heavy metals. R. sphaeroides follows different metabolic strategies when exposed to different stress factors. It is able to regulate its photosynthetic machinery sensing O 2 tensions. When partial O 2 pressure is reduced, R. sphaeroides develops invagination in its cellular membrane. Biosynthesis of chromatophores, which are crucial for photosynthesis, takes place in these invaginations. There are three systems involved in regulation of photosynthetic genes: the PrrBA two subunit activation system, the PpsR repressor and the FnrL, anaerobic regulator. The genes are regulated mainly by O 2 levels. R. sphaeroides has two chromosomes and five naturally occurring plasmids. Many of its genes are duplicated between the two chromosomes. These gene duplications generate the genetic diversity which allows the bacteria widen its spectrum of metabolic capabilities. Chromosome I contains 3106 open reading frames whereas chromosome 2 contains 874. It possesses a flagellum similar to that of E. coli which is able to rotate only in clockwise direction. When exposed to heavy metal stress, R. sphaeroides alters fatty acid composition to increase its membrane integrity. The accumulation of heavy metals in the environments imposes new strategies for restoring the original site conditions. Bioremediation, providing high specificity and versatility has received a great attention in this area. In this study, R. sphaeroides was used to obtain a cobalt-resistant strain. Evolutionary engineering approach of metabolic engineering was used to increase the cobalt tolerance of the bacterium, by growing it under gradually increasing stress conditions. This approach being a 'bottom-up' approach does not require extensive information about the metabolism of the organism. The R. sphaeroides strain R-26 was used in this study. Initial cobalt concentration to be used for selection was determined after screening analysis of the reference strain at 11 different cobalt concentrations. According to the screening results, the selection of the populations was started with 0.1 mM cobalt concentration. Cobalt concentrations were increased gradually from 0.1 mM to 15 mM through 64 generations. Survival rates were monitored every 24 hours by measuring OD 535 values of control and stress cultures. At the same time points, cultures were spread on LB plates to confirm that no contamination has occurred. Cultures which were grown on solid medium were examined morphologically. Cultures that produced carotenoids were identified by their characteristic red colour. When the survival rate decreased to 0.2, the selection process was terminated for further analysis. Growth behaviour of the reference strain and the last population obtained by evolutionary engineering were xixcompared in media with and without cobalt. The cultures showed similar growth characteristics when there was no cobalt in the medium. However, there was a significant difference, when cultures were grown in a medium containing 4 mM cobalt. The 64 th population cells were able to maintain their growth rate nearly the same when exposed to 4 mM cobalt, while the reference strain's growth was inhibited. These results indicate the improved resistance of 64 th population towards cobalt.

Hanay Kamer

İstanbul Technical University · Institute of Graduate Studies in Science

Tersine metabolik mühendislik yöntemi ile aluminyuma dirençli Saccharomyces cerevisiae eldesi

Although Al3+ is non-essential and toxic to life, it is commonly encountered in water, foods, medicines and beverages. Al3+ has been reported to interact with organic molecules in vitro, however, molecular mechanisms of aluminium toxicity/tolerance are not well-known. Aluminium is also related with human neurodegenerative diseases such as Alzheimer and Parkinson. The yeast Saccharomyces cerevisiae is a model organism which has been used widely in molecular biology and biotechnology for a long time. In addition to research, it has also been used for many industrial applications. It can make fermentation which is very important in baking and wine industry. S.cerevisiae can be in haploid and diploid form and its genome has a high degree of homology with those of higher eukaryotes. This makes it important in understanding molecular mechanisms of human-related diseases. In this study, S. cerevisiae was used as a eukaryotic model organism to investigate aluminium tolerance mechanisms. An evolutionary engineering strategy based on batch selection at increasing Al3+ concentrations was employed to obtain aluminium-resistant S. cerevisiae mutants: initially, S. cerevisiae CEN.PK 113-7D wild type strain was chemically mutagenized to increase the genetic diversity of the initial population for selection. 905 (Wild Type) and 906 (EMS mutagenized wild type) were screened under different aluminium levels such as; 0.05, 0.1, 0.4, 0.6, 1, 2, 5, 10, 25, 50 mM to determine the initial aluminium stress level to be used during increasing stress level selections. A batch selection strategy was then applied to obtain aluminium-resistant S. cerevisiae mutants by gradually increasing aluminium-stress levels from 0.5 to 21.5 mM AlCl3 through 43 passages. The 43th last population was spread on plates and 12 individual mutants were randomly chosen. They were tested for their aluminium resistance by using spot assay procedure. According to spot assay results, 4 highly resistant mutants (named as Alu9, Alu10, Alu11, Alu12) were chosen for further analysis. In order to quantify the aluminium resistances of the mutants in detail Most Probable Number method was applied to those 4 mutant individuals. They were then tested for the genetic stability of the Al-resistance.Results showed that the mutants are genetically stable. Cross-resistance tests were also applied using a variety of other metal and non-metal stress types to test if the aluminium-resistant mutants developed cross-resistance or sensitivities to other stress types. For this purpose, the following stresses were tested: 8%(v/v) Ethanol, 0.2 mM NiCl2, 0.7 mM H2O2 , 0.25 mM CuSO4, 1 ml/L phenyl ethanol, 1.3 M MgCl2, 2.5 mM CrCl3, 10 mM MnCl2, 30 mM FeSO4, 80 mM H3BO3, 300 mg/ml Propolis, 0.5 M CaCl2, 2 mM GaNO3, 75 µM AgNO3, 0.20 NiCl2, 10 mM NaCl, 3 mM ZnCl2, 1 mM CoCl2, 2 mM LiCl, pH 4, 25 mM FeCl2. However, no significant cross-resistance or sensitivities to other stress types were detected, except for a slight level of LiCl,GaNO3 and ethanol tolerance, and a slight sensitivity to AgNO3. According to the experimental results, the best individual mutant (Alu10) was chosen for further detailed analyses at physiological and molecular levels.

Saccharomyces cerevisiae

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