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    Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered E. coli
    (Mdpi, 2024) de la Cruz, Mitzi; Kunert, Flavio; Taymaz-Nikerel, Hilal; Sigala, Juan-Carlos; Gosset, Guillermo; Buchs, Jochen; Lara, Alvaro R.
    The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality.
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    Insights Into the Mechanism of Anticancer Drug Imatinib Revealed Through Multi-Omic Analyses in Yeast
    (Mary Ann Liebert, Inc, 2020) Taymaz-Nikerel, Hilal; Eraslan, Serpil; Kirdar, Betul
    Imatinib mesylate is a receptor tyrosine kinase inhibitor drug with broad applications in cancer therapeutics, for example, in chronic myeloid leukemia and gastrointestinal stromal tumors. In this study, new multi-omics findings in yeast on the mechanism of imatinib are reported, using the model organismSaccharomyces cerevisiae. Whole-genome analysis of the transcriptional response of yeast cells following long-term exposure to imatinib, flux-balance analysis (FBA), and modular analysis of protein/protein interaction network consisting of proteins encoded by differentially expressed genes (DEGs) were performed. DEGs indicated that carbon, nitrogen, starch, sucrose, glyoxylate/dicarboxylate metabolism, valine and leucine degradation, and tricarboxylic acid cycle (TCA) were significantly upregulated. By contrast, ribosome biogenesis, pentose/glucuronate interconversion, tryptophan/pyruvate metabolic pathways, and meiosis were significantly downregulated. FBA revealed that a large set of metabolic pathways was altered by imatinib to compensate cancer-associated metabolic changes. Integration of transcriptome and interactome (protein/protein interactions) data helped to identify the core regulatory genes and pathways through elucidation of the active subnetworks. It appears that imatinib may also contribute to antitumoral immune response in the tumor microenvironment and most of the metabolic rearrangements are at posttranscriptional level. Furthermore, additional support for possible contribution of thiamine/pyridoxal phosphate biosynthesis and mitogen-activated protein kinase pathway to drug resistance is noted. This report advances multi-omics understanding of the mechanism of imatinib, and by extension, offers new molecular avenues toward precision medicine and discovery of novel drug targets in cancer therapeutics.
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    Integration of fluxome and transcriptome data in Saccharomyces cerevisiae offers unique features of doxorubicin and imatinib
    (Royal Soc Chemistry, 2021) Taymaz-Nikerel, Hilal
    Improving the efficacy of drugs and developing new drugs are required to compensate for drug resistance. Therefore, it is critical to unveil the mode of action, which can be studied through the cellular response at genome-scale, of the existing drugs. Here, system-level response of Saccharomyces cerevisiae, a eukaryotic model microorganism, to two chemotherapy drugs doxorubicin and imatinib used against cancer are analysed. While doxorubicin is mainly known to interact with DNA through intercalation and imatinib is known to inhibit the activity of the tyrosine kinase enzyme, the exact mechanisms of action for both drugs have not been determined. The response of S. cerevisiae cells to long-term stress by these drugs under controlled aerobic conditions was investigated and analyzed by the genome-wide transcriptome and genome-wide fluxes. The classification of adverse and similar responses of a certain gene at a transcriptional versus flux level indicated the possible regulatory mechanisms under these different stress conditions. Most of the biochemical reactions were found to be regulated at a post-transcriptional or metabolic level, whereas fewer were regulated at a transcriptional level for both stress cases. Furthermore, disparately induced and repressed pathways in the metabolic network under doxorubicin and imatinib stress were identified. The glycolytic and pentose phosphate pathways responded similarly, whereas the purine-histidine metabolic pathways responded differently. Then, a comparison of differential fluxes and differentially co-expressed genes under doxorubicin and imatinib stress provided the potential common and unique features of these drugs. Analyzing such regulatory differences helps in resolving drug mechanisms and suggesting new drug targets.
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    Side effect prediction based on drug-induced gene expression profiles and random forest with iterative feature selection
    (Springernature, 2021) Cakir, Arzu; Tuncer, Melisa; Taymaz-Nikerel, Hilal; Ulucan, Ozlem
    One in every ten drug candidates fail in clinical trials mainly due to efficacy and safety related issues, despite in-depth preclinical testing. Even some of the approved drugs such as chemotherapeutics are notorious for their side effects that are burdensome on patients. In order to pave the way for new therapeutics with more tolerable side effects, the mechanisms underlying side effects need to be fully elucidated. In this work, we addressed the common side effects of chemotherapeutics, namely alopecia, diarrhea and edema. A strategy based on Random Forest algorithm unveiled an expression signature involving 40 genes that predicted these side effects with an accuracy of 89%. We further characterized the resulting signature and its association with the side effects using functional enrichment analysis and protein-protein interaction networks. This work contributes to the ongoing efforts in drug development for early identification of side effects to use the resources more effectively.
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    Time-dependent re-organization of biological processes by the analysis of the dynamic transcriptional response of yeast cells to doxorubicin
    (Royal Soc Chemistry, 2021) Karabekmez, Muhammed Erkan; Taymaz-Nikerel, Hilal; Eraslan, Serpil; Kirdar, Betul
    Doxorubicin is an efficient chemotherapeutic reagent in the treatment of a variety of cancers. However, its underlying molecular mechanism is not fully understood and several severe side effects limit its application. In this study, the dynamic transcriptomic response of Saccharomyces cerevisiae cells to a doxorubicin pulse in a chemostat system was investigated to reveal the underlying molecular mechanism of this drug. The clustering of differentially and significantly expressed genes (DEGs) indicated that the response of yeast cells to doxorubicin is time dependent and may be classified as short-term, mid-term and long-term responses. The cells have started to reorganize their response after the first minute following the injection of the pulse. A modified version of Weighted Gene Co-expression Network Analysis (WGCNA) was used to cluster the positively correlated co-expression profiles, and functional enrichment analysis of these clusters was carried out. DNA replication and DNA repair processes were significantly affected and induced 60 minutes after exposure to doxorubicin. The response to oxidative stress was not identified as a significant term. A transcriptional re-organization of the metabolic pathways seems to be an early event and persists afterwards. The present study reveals for the first time that the RNA surveillance pathway, which is a post-transcriptional regulatory pathway, may be implicated in the short-term reaction of yeast cells to doxorubicin. Integration with regulome revealed the dynamic re-organization of the transcriptomic landscape. Fhl1p, Mbp1p, and Mcm1p were identified as primary regulatory factors responsible for tuning the differentially expressed genes.
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    Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit
    (Wiley, 2024) Lara, Alvaro R.; Kunert, Flavio; Vandenbroucke, Vincent; Taymaz-Nikerel, Hilal; Martinez, Luz Maria; Sigala, Juan-Carlos; Delvigne, Frank
    Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance. The authors propose a strategy that combines cell engineering and genetic circuit design to decouple growth rate from recombinant protein expression based on the carbon source transport. A simple batch scheme allows autoinduction and growth rate shift, mimicking a fed-batch mode. The results are potentially useful for product and process development. image
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    Vitreoscilla haemoglobin: A tool to reduce overflow metabolism
    (MDPI, 2022-01) Taymaz-Nikerel, Hilal
    Abstract: Overflow metabolism is a phenomenon extended in nature, ranging from microbial to cancer cells. Accumulation of overflow metabolites pose a challenge for large-scale bioprocesses. Yet, the causes of overflow metabolism are not fully clarified. In this work, the underlying mechanisms, reasons and consequences of overflow metabolism in different organisms have been summarized. The reported effect of aerobic expression of Vitreoscilla haemoglobin (VHb) in different organisms are revised. The use of VHb to reduce overflow metabolism is proposed and studied through flux balance analysis in E. coli at a fixed maximum substrate and oxygen uptake rates. Simulations showed that the presence of VHb increases the growth rate, while decreasing acetate production, in line with the experimental measurements. Therefore, aerobic VHb expression is considered a potential tool to reduce overflow metabolism in cells. © 2021 by the authors.