| 1. |
- Cámara, Elena, 1985, et al.
(författare)
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Novel methods for accelerating the development of more inhibitor tolerant yeast strains for cellulosic ethanol production
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Production of fuels and chemicals from biomass is a crucial step towards a society not depending on fossil resources. Second generation bioethanol, with lignocellulose material as feedstock, is a promising alternative to first generation bioethanol produced from sugar-based raw materials. Still, in order for biofuels to substitute for fossil based fuels the production needs to be significantly more efficient and price competitive. One way to tackle the suboptimal productivity is to develop production hosts with increased tolerance towards inhibitors found in lignocellulosic hydrolysates. Our focus is on accelerating the design-build-test-learn cycle for making industrial yeast strains for conversion of lignocellulosic biomass. An efficient, marker-free genome editing strategy for engineering polyploid strains is needed for engineering the robustness of industrial yeasts. Here, we combine CRISPR/Cas9 technologies for strain engineering with high-throughput strain analysis using microbioreactors. We have developed a method to study hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates. This way, we can accelerate the development of more robust production hosts as well as gain novel understanding on microbial physiology.
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| 2. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2018
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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 biochemical with its renewable counterpart requires the production to be significantly more efficient and price competitive. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulose hydrolysate. 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. Numerous attempts have been reported to improve the stress tolerance of Saccharomyces cerevisiae by different bioengineering strategies such as deletion/overexpression of genes. However, the inability to achieve a fine balance of the transcriptional expression of the target and the ancillary gene(s) is one of the major factors that impedes the efficiency of many of these strategies. In this project, we apply CRISPR interference (CRISPRi) technology to investigate the potential of fine-tuning the expression of genes that are related to the stress regulation. 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 a sgRNA. Strains with altered regulation will be screened for inhibitor tolerance. Furthermore, transcriptomics analysis of tolerant mutants will be conducted 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. Here, we will show data on a methodology that we have developed for studying hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates.
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| 3. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Small scale screening of yeast strains enables high-throughput evaluation of performance in lignocellulose hydrolysates
- 2020
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Ingår i: Bioresource Technology Reports. - : Elsevier BV. - 2589-014X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Second generation biorefineries demand efficient lignocellulosic hydrolysate fermenting strains and recent advances in strain isolation and engineering have progressed the bottleneck in developing production hosts from generation of strains into testing these under relevant conditions. In this paper, we introduce a methodology for high-throughput analysis of yeast strains directly in lignocellulosic hydrolysates. The Biolector platform was used to assess aerobic and anaerobic growth of 12 Saccharomyces cerevisiae strains and their ΔPdr12 mutants in wheat straw hydrolysate. The strains evaluated included lab, industrial and wild type strains and the screening could capture significant differences in growth and ethanol production among the strains. The methodology was also demonstrated with corn stover hydrolysate and the results were in line with shake flask cultures. Our study demonstrates that growth in lignocellulosic hydrolysates could be rapidly monitored using 1 ml cultures and that measuring growth and product formation under relevant conditions are crucial for evaluating strain performance.
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