Project description:The environmental stresses and inhibitors encounted by Saccharomyces cerevisiae strains are main limiting factors in bioethanol fermentation. Investigation of the molecular mechanisms underlying the stresses-related phenotypes diversities within and between S. cerevisiae populations could guide the construction of yeast strains with improved stresses tolerance and fermentation performances. Here, we explored the genetic characteristics of the bioethanol S. cerevisiae strains, and elucidated the genetic variations correlated with its advantaged traits (higher ethanol yield under sever conditions and better tolerance to multiple stresses compared to an S288c derived laboratory strain BYZ1). Firstly, pulse-field gel electrophoresis combined with array-comparative genomic hybridization was used to compare the genome structure of industrial strains and the laboratory strain BYZ1.
Project description:Natural S. cerevisiae isolates were evaluated for tolerance to a complex mixture of lignocellulosic inhibitors. Two isolates, with intermediate and superior tolerance characteristics, were investigated to determine the proteomic changes required for tolerance to lignocellulosic inhibitors.
Project description:The oleaginous red yeast species Rhodotorula toruloides, a prominent environmental basidiomycetous yeast, has garnered significant interest for its remarkable capacity to utilize main carbon sources present in lignocellulosic hydrolysates, such as glucose, xylose, and acetic acid, and to efficiently produce lipids and carotenoids. This species can also efficiently use other less usual and difficult-to-catabolize C-sources, as is the case of the acid sugar D-galacturonic acid and the neutral sugar L-arabinose, present in hydrolysates from pectin-rich agro-industrial residues. Strain R. toruloides IST536 (alias PYCC 5615) was previously selected in our laboratory for sugar beet pulp valorization based on its ability to produce lipids and carotenoids through the complete catabolism of the major C-sources present in the hydrolysates (1). This strain, whose genome has been recently sequenced in our laboratory, is a conjugated strain derived from IFO 0559 (isolated from wood-pulp; GCA_000988805.1) × IFO 0880 (isolated from air; GCA_001255795.1) (2). Despite its potential, the metabolism of this promising strain can be limited by the presence of acetic acid in the hydrolysates as well as other inhibitors present in lignocellulosic hydrolysates. To increase IST536 (PYCC 5615) strain robustness, an adaptive laboratory evolution (ALE) strategy was used. The selected evolved strain IST536 MM15 exhibits increased tolerance to several inhibitors of biotechnological relevance: methanol and the four main inhibitors present in lignocellulosic hydrolysates: acetic and formic acids, hydroxymethylfurfural (HMF), and furfural (3). The superior performance of this evolved multi-stress tolerant strain for lipid production from non-detoxified lignocellulosic biomass hydrolysates was confirmed. To obtain mechanistic insights underlying such multi-tolerant phenotype, the genomes of the original and the evolved strains, IST536 (PYCC 5615) and IST536 MM15, respectively, were sequenced and the transcriptomic profiling of both strains under non-stressing conditions was performed. References: 1. Martins LC, et, al. Journal of Fungi. 2021; 7(3):215. https://doi.org/10.3390/jof7030215 2. Banno, I. (1967). The Journal of General and Applied Microbiology, 13(2), 167-196. https://doi.org/10.2323/jgam.13.167 3. Fernandes, M. A., Mota, M. N., et al, Journal of Fungi, 2023, 9(11), 1073 https://doi.org/10.3390/jof9111073
Project description:The environmental stresses and inhibitors encounted by Saccharomyces cerevisiae strains are main limiting factors in bioethanol fermentation. Investigation of the molecular mechanisms underlying the stresses-related phenotypes diversities within and between S. cerevisiae populations could guide the construction of yeast strains with improved stresses tolerance and fermentation performances. Here, we explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329, and elucidated the genetic variations correlated with its advantaged traits (higher ethanol yield under sever conditions and better tolerance to multiple stresses compared to an S288c derived laboratory strain BYZ1). Firstly, pulse-field gel electrophoresis combined with array-comparative genomic hybridization was used to compare the genome structure of YJS329 and the laboratory strain BYZ1. Yeast cells were cultured in YPD medium. Genome DNA of YJS329 and BYZ1 was isolated and sonicated. The average length of DNA fragments was 200-1000bp. The shearing DNA was labeled with Cy5/Cy3 and hybridized to NimbleGen S.cerevisiae Whole-Genome Tiling arrays, which is single array design containing all chromosomes with 32bp median probe spacing and totally covered by ~385,000 probes. Scanning was performed with the Axon GenePix 4000B microarray scanner.
Project description:Xylose-utilizing yeasts with tolerances to fermentation inhibitors (such as weak organic acids) and high temperature are needed for cost-effective simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic materials. We constructed a novel xylose-assimilating Saccharomyces cerevisiae strain with improved fermentation performance under heat and acid co-stress using the genome shuffling technique. Two xylose-utilizing diploid yeasts with different genetic backgrounds were used as the parental strains for genome shuffling. The hybrid strain Hyb-8 showed significantly higher xylose fermentation ability than both parental strains (Sun049T-Z and Sun224T-K) under co-stress conditions of heat and acids. To screen for genes that might be important for fermentation under heat and acid co-stress, a transcriptomic analysis of hybrid strain Hyb-8 and its parental strains was performed.
Project description:Thermotolerance development of robust Saccharomyces cerevisiae is necessary to enhance enzyme activity of cellulase, lower cooling costs, and reduce cell harm from the bad-distributed heat transfer in large-scale fermentation. The process-based studies of adaptive evolution have been well documented, but it remains unknown for the underlying molecular mechanism of the improved thermotolerance and the facilitated ethanol fermentability derived from adaptive evolution. Here, a robust thermotolerant S. cerevisiae Z100 was obtained with significantly improved ethanol fermentability under the stress of high temperature (50 oC) after 91 days’ adaptive evolution. RNA sequencing showed that adaptive evolution and its derived thermotolerance contributed to the unique gene transcriptional landscapes of the evolved strain. An interesting phenomenon was that the gene transcriptional signals of carbon metabolism were strengthened not at 50 oC but at 30 oC in S. cerevisiae Z100, and thus suggested that the improved thermotolerance led to the enhanced ethanol fermentability at 30 oC. The deeply repressed gene transcriptional expression indicated ribosome would be another key thermotolerant mechanism for the evolved strain. This study would provide a robust thermotolerant S. cerevisiae for bioethanol production and an important clue for future synthetic biology to thermotolerance engineering of fermentation strains.
Project description:Adaptive evolution experiment for enhaced tolerance to hydrolysates of lignocellulosic biomass in S. cerevisiae. The samples involves a batch culture in YNB and Hydrolysates. Cells were harvested at mid-exponential phase.
Project description:Resistance of Saccharomyces cerevisiae to high furfural concentration is based on NADPH-dependent reduction by at least two oxireductases. Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural resistant strain and its parent in micro-aerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower furfural concentrations, NADH-dependent oxireductases are the main defence mechanism. At concentrations above 15 mM, however, [1-13C]-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through pentose-phosphate pathway increases and that NADPH-dependent oxireductases became the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is up-regulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterised ORF YKL071W and 4 further, likely NADPH-dependent oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inverse engineered significantly increased furfural resistance in the parent strain, thereby demonstrating these two enzymes to be key elements of the resistance phenotype. Experiment Overall Design: RNA levels were measured in glucose limited, micro-aerobic chemostat cultures with different concentrations of the growth inhibitor furfural. Two strains were compared: TMB3400-FT30-3 is a strain that has been evolutionary adapted to withstand high furfural concentrations. TMB3400 is its less resistant parent. Number of biological replicates: 2-3.
Project description:Lignocellulosic biomass is an abundant renewable resource with tremendous potential to alleviate climate crisis. Yarrowia lipolytica is an attractive biochemical production host, while the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate restricts the efficient utilization of this resource. Given a deficient understanding of the inherent interactions between these inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using CRISPR interference library in Y. lipolytica, achieving tolerance to 0.35% (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15% (v/v) acetic acid. The tolerance mechanism might involve improvements of signal transduction, PP pathway, and TCA cycle. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for significantly improving microbial tolerance to inhibitors in lignocellulosic hydrolysate and profoundly revealing underlying mechanism.