Exploring Saccharomyces cerevisiae’s responses to acetic acid and other inhibitors found in lignocellulosic hydrolysates
Doctoral thesis, 2023
The limited tolerance of the budding yeast Saccharomyces cerevisiae to lignocellulosic hydrolysate inhibitors is a key challenge to its use in biorefinery cell factories. Considerable resources have been invested in the isolation of yeast strains with better tolerance towards the inhibitors released during lignocellulose hydrolysis, such as acetic acid. The goal of this thesis was twofold: characterize the transcriptional response of S. cerevisiae to wheat straw hydrolysate and explore the role of essential S. cerevisiae genes in acetic and formic acid tolerance, using a new biosensor and competitive growth assays.
The transcriptomes of one laboratory strain, two industrial strains, and two wild-type isolates grown in wheat straw hydrolysate were profiled. Despite similar growth, the isolates showed different expression of genes encoding proteins involved in oxidative stress response, lipid accumulation, and transport, suggesting different genetic strategies for tolerance. The new acetic acid biosensor was based on two transcription factors, Haa1 from S. cerevisiae and BM3R1 from Bacillus megaterium. Biosensor and competitive growth were used in parallel to screen a S. cerevisiae CRISPR interference strain library. While fluorescence-activated cell sorting led to the isolation of cells with higher acetic acid retention and sensitivity, competitive growth assays allowed the identification of cells with higher acid tolerance. The results confirmed the role in acid stress response of genes involved in glycogen accumulation, chromatin modification, and mitochondrial or proteasomal functions. Two novel targets for improving tolerance were also identified: PAP1 and HIP1.
Altogether, this thesis provides mechanistic insight into the stress response to lignocellulosic hydrolysates or weak acid inhibitors that limit yeast-mediated conversion of lignocellulosic biomass into biochemicals. Additionally, it offers new tools for the identification of strains with altered acetic acid tolerance.
The transcriptomes of one laboratory strain, two industrial strains, and two wild-type isolates grown in wheat straw hydrolysate were profiled. Despite similar growth, the isolates showed different expression of genes encoding proteins involved in oxidative stress response, lipid accumulation, and transport, suggesting different genetic strategies for tolerance. The new acetic acid biosensor was based on two transcription factors, Haa1 from S. cerevisiae and BM3R1 from Bacillus megaterium. Biosensor and competitive growth were used in parallel to screen a S. cerevisiae CRISPR interference strain library. While fluorescence-activated cell sorting led to the isolation of cells with higher acetic acid retention and sensitivity, competitive growth assays allowed the identification of cells with higher acid tolerance. The results confirmed the role in acid stress response of genes involved in glycogen accumulation, chromatin modification, and mitochondrial or proteasomal functions. Two novel targets for improving tolerance were also identified: PAP1 and HIP1.
Altogether, this thesis provides mechanistic insight into the stress response to lignocellulosic hydrolysates or weak acid inhibitors that limit yeast-mediated conversion of lignocellulosic biomass into biochemicals. Additionally, it offers new tools for the identification of strains with altered acetic acid tolerance.
acetic acid biosensor
formic acid
CRISPRi screening
acetic acid
Saccharomyces cerevisiae
lignocellulosic inhibitor tolerance
differential gene expression
Author
Maurizio Mormino
Chalmers, Life Sciences, Industrial Biotechnology
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
Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae
FEMS Yeast Research,;Vol. 21(2021)
Journal article
Lenitz I., Mormino M., Blomberg A., Mukherjee V., Nygård Y. Pooled CRISPRi screen of essential genes of Saccharomyces cerevisiae reveals genes important for tolerance to acetic acid and formic acid
Cámara E.*, Mormino M.*, Siewers V., Nygård Y. Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show great differences in transcriptional stress responses
Understanding how yeast responds to stress can allow for greener industrial production
The transition towards a greener society that is less reliant on petroleum, is a central priority for mankind survival. Genetically modified yeasts can nowadays be used to produce a wide spectrum of chemicals and therefore represent a competitive alternative to fossil-based production. An advantage with yeasts is that they can grow on agricultural and forestry residual products to produce various useful chemicals. However, the residual products need to be pretreated to release energy (sugars) that yeasts can use. This unfortunately releases toxic compounds which hamper yeast productivity.
In this thesis I investigate how yeast reacts to the stress caused by these toxic compounds. First, I explore how different yeasts respond to being cultivated on a specific forestry residual product, previously pretreated. Then, I focus on two of the most abundant inhibitors found in the forestry residual products after the pretreatments: acetic acid and formic acid. For this, I developed and used a novel biological tool for acetic acid sensing, which allows to detect and report the acetic acid produced or accumulated inside the cells. I also analyzed genes that are involved in yeast adaptation to acetic and formic acid. Altogether, this thesis provides crucial insights into yeast responses to stress that is present in yeast-based biorefineries. This knowledge can be used to develop modified yeasts with improved performances and tolerance, thus contributing to a greener industrial production.
The transition towards a greener society that is less reliant on petroleum, is a central priority for mankind survival. Genetically modified yeasts can nowadays be used to produce a wide spectrum of chemicals and therefore represent a competitive alternative to fossil-based production. An advantage with yeasts is that they can grow on agricultural and forestry residual products to produce various useful chemicals. However, the residual products need to be pretreated to release energy (sugars) that yeasts can use. This unfortunately releases toxic compounds which hamper yeast productivity.
In this thesis I investigate how yeast reacts to the stress caused by these toxic compounds. First, I explore how different yeasts respond to being cultivated on a specific forestry residual product, previously pretreated. Then, I focus on two of the most abundant inhibitors found in the forestry residual products after the pretreatments: acetic acid and formic acid. For this, I developed and used a novel biological tool for acetic acid sensing, which allows to detect and report the acetic acid produced or accumulated inside the cells. I also analyzed genes that are involved in yeast adaptation to acetic and formic acid. Altogether, this thesis provides crucial insights into yeast responses to stress that is present in yeast-based biorefineries. This knowledge can be used to develop modified yeasts with improved performances and tolerance, thus contributing to a greener industrial production.
Driving Forces
Sustainable development
Subject Categories
Industrial Biotechnology
Biochemistry and Molecular Biology
Areas of Advance
Life Science Engineering (2010-2018)
ISBN
978-91-7905-809-8
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5275
Publisher
Chalmers