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- Torello Pianale, Luca, 1995, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil-based chemicals with renewable biochemicals requires the production to be significantly more efficient and price competitive. Remediation of several technical bottlenecks is needed before this can be accomplished. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulosic hydrolysates. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. In this project, we are trying to fine tune the expression of stress related genes to boost the stress tolerance in Saccharomyces cerevisiae using the CRISPR interference (CRISPRi) technology. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using an sgRNA. Using a high-throughput yeast transformation method developed in our laboratory, we are generating a CRISPRi strain library. Each strain in this library has altered regulation for at-least one stress related gene. Next, high-throughput phenotypic evaluation of this library is performed by growing the strains under the exposure of inhibitors relevant to lignocellulosic hydrolysates. Here, we will demonstrate our primary CRISPRi library data. Further, we will explain the high-throughput methodologies for generating the CRISPRi mutants and to study their hydrolysate tolerance, adaptation and ethanol production capacity at microscale. In future, we will perform transcriptomics analysis of the most tolerant mutants to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate.
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| 2. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
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Phenomics, transcriptomics and metabolomics for identifying concentration-dependent chemical interactions and understanding the mechanistic basis of the mixture toxicity
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The prevalence of mixtures of synthetic and natural chemicals in the environment is a growing concern for public health and environmental effects. Currently, most chemical legislations are based on the risk assessments carried out on individual substances and theoretical estimates of combination effect. However, exposure to multi-component mixtures may stimulate unpredicted overall toxic responses due to interactions, where interactions were scored as deviations from the independent action model. In our project, we investigated the frequency and magnitude of interactions in mixtures of five compounds - NaCl, HgCl2, paraquat, rapamycin, clotrimazole - with relatively known specific mode of action. Growth effects by all-combination pair-wise mixtures spanning a wide concentration range were investigated by employing high-resolution yeast phenomics. The baker’s/brewer’s yeast Saccharomyces cerevisiae and the marine yeast Debaryomyces hansenii are used in this study to identify evolutionary conserved mixture effects, with the aim to identify generic responses of relevance to a vast array of organisms. Our results clearly show that both synergistic and antagonistic relationships exist among the tested chemicals and some of these relationships are concentration-dependent. Evolutionary conserved interactions on the level of rate of growth were found for salt and rapamycin (synergy) as well as for salt and paraquat (antagonism). The mechanistic basis of the chemical interactions identified in our study was investigated by transcriptomics and metabolomics. As one example, we observed that several genes with symporter activity and with cation transmembrane transporter activity is downregulated in salt plus paraquat mixtures, while the expression of genes that are related to cofactor-dependent metabolic pathways is stimulated. We believe that the repression of symporter and ion transmembrane transport activity reduces paraquat entry to the yeast cells and thereby reduces its toxic response when combined with salt. On the other hand, upregulation of several of the genes (such as PGI1, PFK1, FBA1, and CDC19) related to cofactor-dependent metabolic pathways boost yeast fermentative activity. Since paraquat induces the production of reactive oxygen species (ROS) via respiration, a shift from aerobic respiration to anaerobic fermentation can reduce formation of ROS, thus reduces oxidative stress by paraquat.
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