| 1. |
- Cámara, Elena, 1985, et al.
(författare)
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A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to similar to 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.
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| 2. |
- Cámara, Elena, 1985, et al.
(författare)
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CRISPR interference technology for development of more tolerant industrial yeast strains (Milan)
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Second generation bioethanol using lignocellulosic biomass as raw material is a promising alternative to bioethanol produced from sugar-based feedstocks. In addition to sugars, lignocellulosic hydrolysates also contain inhibitors that impair microbial growth. One way to tackle the low productivities is to develop new strains with increased tolerance towards inhibitors. Over the past few years, different CRISPR technologies have been developed to accelerate the construction of new strains. The CRISPR interference (CRISPRi) technology utilizes a catalytically inactive Cas9 (dCas9) to modulate the expression of genes targeted by a sgRNA, allowing the alteration of essential genes and the manipulation of multiple traits without altering the target sequence. In the present work, our goal was to use CRISPRi to improve the inhibitor tolerance of a polyploid industrial yeast strain. We explored different strategies to overcome the challenges of implementing CRISPRi in an industrial strain. As a proof of concept, the expression of a gene encoding a fluorescent protein was modulated using dCas9 with different activation or repression domains. Changes in fluorescence were measured by flow cytometry and changes in expression were verified by qPCR, validating the use of CRISPRi for alteration of gene expression in an industrial yeast strain. Subsequently, a number of genes previously identified to be involved in inhibitor tolerance were selected as targets for CRISPRi. The performance of the novel strains during growth in the presence of different inhibitors was analysed in a high-throughput platform, leading to identification of strains where the altered gene expression led to improved tolerance. This work shows that the CRISPRi technology can be used to accelerate the development of more robust, industrial production hosts.
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| 3. |
- Cámara, Elena, 1985, et al.
(författare)
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Data mining of Saccharomyces cerevisiae mutants engineered for increased tolerance towards inhibitors in lignocellulosic hydrolysates
- 2022
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 57
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Forskningsöversikt (refereegranskat)abstract
- The use of renewable plant biomass, lignocellulose, to produce biofuels and biochemicals using microbial cell factories plays a fundamental role in the future bioeconomy. The development of cell factories capable of efficiently fermenting complex biomass streams will improve the cost-effectiveness of microbial conversion processes. At present, inhibitory compounds found in hydrolysates of lignocellulosic biomass substantially influence the performance of a cell factory and the economic feasibility of lignocellulosic biofuels and chemicals. Here, we present and statistically analyze data on Saccharomyces cerevisiae mutants engineered for altered tolerance towards the most common inhibitors found in lignocellulosic hydrolysates: acetic acid, formic acid, furans, and phenolic compounds. We collected data from 7971 experiments including single overexpression or deletion of 3955 unique genes. The mutants included in the analysis had been shown to display increased or decreased tolerance to individual inhibitors or combinations of inhibitors found in lignocellulosic hydrolysates. Moreover, the data included mutants grown on synthetic hydrolysates, in which inhibitors were added at concentrations that mimicked those of lignocellulosic hydrolysates. Genetic engineering aimed at improving inhibitor or hydrolysate tolerance was shown to alter the specific growth rate or length of the lag phase, cell viability, and vitality, block fermentation, and decrease product yield. Different aspects of strain engineering aimed at improving hydrolysate tolerance, such as choice of strain and experimental set-up are discussed and put in relation to their biological relevance. While successful genetic engineering is often strain and condition dependent, we highlight the conserved role of regulators, transporters, and detoxifying enzymes in inhibitor tolerance. The compiled meta-analysis can guide future engineering attempts and aid the development of more efficient cell factories for the conversion of lignocellulosic biomass.
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| 4. |
- Cámara, Elena, 1985, et al.
(författare)
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Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
- 2019
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 3 February:https://doi.org/10.1002/bit.26947
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Tidskriftsartikel (refereegranskat)abstract
- The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn. Komagataella spp.) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.
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| 5. |
- 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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| 6. |
- Cámara, Elena, 1985, et al.
(författare)
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Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses
- 2024
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 90:5
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Tidskriftsartikel (refereegranskat)abstract
- Improving our understanding of the transcriptional changes of Saccharomyces cerevisiae during fermentation of lignocellulosic hydrolysates is crucial for the creation of more efficient strains to be used in biorefineries. We performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. Many of the differently expressed genes identified among the strains have previously been reported to be important for tolerance to lignocellulosic hydrolysates or inhibitors therein. Our study demonstrates that stress responses typically identified during aerobic conditions such as glutathione metabolism, osmotolerance, and detoxification processes also are important for anaerobic processes. Overall, the transcriptomic responses were largely strain dependent, and we focused our study on similarities and differences in the transcriptomes of the LBCM strains. The expression of sugar transporter-encoding genes was higher in LBCM31 compared with LBCM109 that showed high expression of genes involved in iron metabolism and genes promoting the accumulation of sphingolipids, phospholipids, and ergosterol. These results highlight different evolutionary adaptations enabling S. cerevisiae to strive in lignocellulosic hydrolysates and suggest novel gene targets for improving fermentation performance and robustness.
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| 7. |
- 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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| 8. |
- 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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