A cRiSpR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12 elena cámara, ibai Lenitz & Yvonne nygård * 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 ~ 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. The yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites 1,2. However, some of the current production processes are compromised by low yields and productivities, thus further optimization is required 3. In particular, the production of second-generation bioethanol and other biochemicals from lignocellulosic biomass, which provides an alternative to oil-based chemicals, suffers from sub-optimal productivity 4. During the hydrolysis of the raw material, inhibitory compounds (e.g. organic acids and aromatic aldehydes) are formed or released, compromising the microbial performance 5. While quite some work has been done on elucidating genes required for tolerance, much less work has been done on improving tolerance towards stress by altering expression of genes 6. Some previous studies demonstrated deletion 7-9 or overexpression 10-14 of endogenous genes to improve tolerance of S. cerevisiae towards inhibitors commonly found in lignocellulosic hydrolysates. However, most of the published work focuses on improving the tolerance of laboratory yeast strains that generally have weaker tolerance to stress 6 , while translation of beneficial modifications to more robust, industrial strains often is very challenging. The choice of yeast strain to be engineered is crucial for the successful implementation of the engineered phenotype in an industrial production process 15. Yeast strains used in industrial processes tend to be genetically diverse, since they usually arise from hybridization between different species 16. Hence...
Background Acetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response. Results An acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1. Conclusions This study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.