CRISPRi/a for investigating yeast tolerance to inhibitors in lignocellulosic hydrolysates
Doctoral thesis, 2023
Saccharomyces cerevisiae has immense potential as a cell factory in various biotechnological processes where biomass from agricultural industry residues is used as feedstock. Nonetheless, the inhibitors released during the pretreatment of the biomass makes lignocellulosic hydrolysates a challenging substrate for microorganisms. In this thesis, the CRISPR interference/activation (CRISPRi/a) technology was used in combination with high-throughput screening methods to improve tolerance of S. cerevisiae towards inhibitors found in lignocellulosic hydrolysates. The focus was on understanding the genetics behind formic and acetic acid tolerance, two abundant inhibitory compounds. The aims were to compare the responses to either acid and to explore how the results could be extrapolated to understand hydrolysate tolerance.
The CRISPRi/a technology was used to improve the hydrolysate tolerance of an industrial strain and to alter the expression of the transcription factor encoding genes YAP1 and PDR1, leading to strains with altered tolerance to acetic acid. We performed ChIP-exo experiments, which demonstrated that both transcription factors showed increased binding of target genes in the presence of acetic acid. Notably, genes related to amino acid synthesis and cell membrane transporters were highly bound to Yap1 and Pdr1 in the presence of acetic acid. Furthermore, A CRISPRi strain library targeting the essential and respiratory essential genes in S. cerevisiae was studied for acetic and formic acid tolerance. The strains were screened by using various high-throughput methods such as competitive growth assays, fluorescence-activated cell sorting and screening for growth on solid media. Systematic analysis of the data highlighted genes encoding proteins with functions in intracellular vesicle transport, glycogen accumulation or chromatin regulation as important for tolerance towards acetic and formic acid. Interesting strains were further characterized individually in the presence of acetic or formic acid, in a synthetic hydrolysate medium or in the presence of oxidative stress causing agents.
To conclude, this research advances our knowledge on how the regulation of genes such as the ones related to chromatin remodeling can influence tolerance to weak acids as well as other inhibitors found in lignocellulosic hydrolysates. The results demonstrate the potential of CRISPRi/a technology to accelerate the development of more tolerant industrial yeast strains.
The CRISPRi/a technology was used to improve the hydrolysate tolerance of an industrial strain and to alter the expression of the transcription factor encoding genes YAP1 and PDR1, leading to strains with altered tolerance to acetic acid. We performed ChIP-exo experiments, which demonstrated that both transcription factors showed increased binding of target genes in the presence of acetic acid. Notably, genes related to amino acid synthesis and cell membrane transporters were highly bound to Yap1 and Pdr1 in the presence of acetic acid. Furthermore, A CRISPRi strain library targeting the essential and respiratory essential genes in S. cerevisiae was studied for acetic and formic acid tolerance. The strains were screened by using various high-throughput methods such as competitive growth assays, fluorescence-activated cell sorting and screening for growth on solid media. Systematic analysis of the data highlighted genes encoding proteins with functions in intracellular vesicle transport, glycogen accumulation or chromatin regulation as important for tolerance towards acetic and formic acid. Interesting strains were further characterized individually in the presence of acetic or formic acid, in a synthetic hydrolysate medium or in the presence of oxidative stress causing agents.
To conclude, this research advances our knowledge on how the regulation of genes such as the ones related to chromatin remodeling can influence tolerance to weak acids as well as other inhibitors found in lignocellulosic hydrolysates. The results demonstrate the potential of CRISPRi/a technology to accelerate the development of more tolerant industrial yeast strains.
Biosensor
Screening
Yeast
CRISPRi/a
Competitive growth assay
Acetic acid
ChIP-exo
Lignocellulosic hydrolysates
Formic acid
Tolerance
10'an,, Chemistry Building floor 10, Kemigården 4
Opponent: Uffe Hasbro Mortensen, Technical University of Denmark
Author
Ibai Lenitz Etxaburu
Chalmers, Life Sciences, Industrial Biotechnology
A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12
Scientific Reports,;Vol. 10(2020)
Journal article
Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library
Microbial Cell Factories,;Vol. 21(2022)p. 214-
Journal article
CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae
Engineering Microbiology,;Vol. 3(2023)
Journal article
Lenitz, I., Mormino, M., Blomberg, A., Mukherjee, V., Nygård, Y. (2023) Pooled CRISPRi screen of essential genes of Saccharomyces cerevisiae reveals genes important for tolerance to acetic acid and formic acid
ChIP-exo and CRISPRi/a illuminate the role of Pdr1 and Yap1 in acetic acid tolerance in <i>Saccharomyces cerevisiae</i>
Applied and Environmental Microbiology,;Vol. 91(2025)
Journal article
As awareness towards the consequences of climate change has increased, governments and industry have increased their support and efforts to use renewable sources to produce commodity chemicals. In this context, development of cell factories to produce chemicals from plant biomass has gained increasing relevance and attention. One important host in cell factories is Saccharomyces cerevisiae, a yeast that has been historically used for the production of bread, wine and beer. S. cerevisiae is a robust yeast that is well suited for industrial production. Moreover, it has been studied in depth and is therefore a model organism with a well-developed genetic toolbox.
Plant biomass, which is also referred to as lignocellulosic biomass, is composed of three main fractions: cellulose, hemicellulose and lignin. The combination of these three fractions creates a highly stable structure that needs to be pretreated to release the sugars that yeast can utilize. The pretreatment unfortunately releases different inhibitory by-products which reduce yeast productivity. This represents a significant challenge in the field.
In my thesis work, I altered the gene expression of S. cerevisiae to obtain strains that are better suited for conversion of lignocellulosic biomass. Concretely, I explored ways to make yeast more tolerant towards inhibitors found in lignocellulosic biomass and contributed to broadening the knowledge about various tolerance related genes in yeast. In my work, I used genetic engineering tools to modulate the transcription yeast genes that are related to cell stress. I tested the efficacy of tolerance engineering in different S.cerevisiae strain backgrounds; one used in research and the other one in specialized in industrial ethanol production. Furthermore, I utilized and compared different high-throughput screening methods to investigate inhibitor tolerance of strain libraries comprising of thousands of strains at the same time. As a result, many gene targets that could be utilized in the future development of cell factories were identified.
My thesis work has contributed to the collective understanding on tolerance mechanisms of yeast against inhibitors found in plant biomass, as well as established new screening methods that allow us to research tolerance in a faster, more efficient way. The knowledge and insights obtained in this thesis can therefore contribute to the development of next generation yeast cell factories used in renewable production of biochemicals.
Plant biomass, which is also referred to as lignocellulosic biomass, is composed of three main fractions: cellulose, hemicellulose and lignin. The combination of these three fractions creates a highly stable structure that needs to be pretreated to release the sugars that yeast can utilize. The pretreatment unfortunately releases different inhibitory by-products which reduce yeast productivity. This represents a significant challenge in the field.
In my thesis work, I altered the gene expression of S. cerevisiae to obtain strains that are better suited for conversion of lignocellulosic biomass. Concretely, I explored ways to make yeast more tolerant towards inhibitors found in lignocellulosic biomass and contributed to broadening the knowledge about various tolerance related genes in yeast. In my work, I used genetic engineering tools to modulate the transcription yeast genes that are related to cell stress. I tested the efficacy of tolerance engineering in different S.cerevisiae strain backgrounds; one used in research and the other one in specialized in industrial ethanol production. Furthermore, I utilized and compared different high-throughput screening methods to investigate inhibitor tolerance of strain libraries comprising of thousands of strains at the same time. As a result, many gene targets that could be utilized in the future development of cell factories were identified.
My thesis work has contributed to the collective understanding on tolerance mechanisms of yeast against inhibitors found in plant biomass, as well as established new screening methods that allow us to research tolerance in a faster, more efficient way. The knowledge and insights obtained in this thesis can therefore contribute to the development of next generation yeast cell factories used in renewable production of biochemicals.
Subject Categories (SSIF 2011)
Biochemistry and Molecular Biology
Microbiology
Bioinformatics and Systems Biology
Biocatalysis and Enzyme Technology
ISBN
978-91-7905-910-1
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5376
Publisher
Chalmers
10'an,, Chemistry Building floor 10, Kemigården 4
Opponent: Uffe Hasbro Mortensen, Technical University of Denmark