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- 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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| 2. |
- 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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| 3. |
- 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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