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The enzymatic and non-enzymatic defense systems of oxidative stress response for the adapted industrial yeast S. cerevisiae NRRL Y-50049 against synergistic challenge of furfural and HMF. F-H ROS stands for furfural-HMF caused reactive oxygen species. Enzymatic defense system is boxed in gray and the non-enzymatic defense system in blank. Genes showing significantly enhanced expressions are bolded and key genes underlined
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The industrial yeast Saccharomyces cerevisiae has a plastic genome with a great flexibility in adaptation to varied conditions of nutrition, temperature, chemistry, osmolarity, and pH in diversified applications. A tolerant strain against 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) was successfully obtained previously by adapta...
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Citations
... In addition, a chitin synthase encoding gene (CHS1) has been associated to acetic and ethanol tolerance, and MNN2, encoding a α-mannosyltransferase was linked to acetic acid stress [50]. This cell wall remodeling response appears essential for limiting the entry of toxic compounds and enhancing overall cellular robustness against diverse stress effectors, including chemical inhibitors present in lignocellulosic hydrolysates, such as organic acids and furan aldehydes [32]. The dynamic remodelling of the cell wall also facilitates cellular adaptation to fluctuating external osmolarities [50]. ...
Background
The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.
Results
Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.
Conclusions
The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.
... The study showed the importance of in situ detoxification of these inhibitors by the inhibitor-tolerant, evolved yeast strains for second-generation bioethanol production [109]. In a later study, Liu and Ma [110] also investigated the transcriptomic responses of a furfural and HMF-tolerant, evolved strain, upon exposure to these inhibitors. The comparative transcriptomic analysis results revealed some key pathways such as the cell wall response, endogenous and exogenous cellular detoxification pathways and specific transcription factors like Yap1, Met4, Msn2/4 and Pdr1/3 as the main differentiated components of the inhibitortolerant strain, which may have a role in the complex genetics of HMF and furfural tolerance [110]. ...
... In a later study, Liu and Ma [110] also investigated the transcriptomic responses of a furfural and HMF-tolerant, evolved strain, upon exposure to these inhibitors. The comparative transcriptomic analysis results revealed some key pathways such as the cell wall response, endogenous and exogenous cellular detoxification pathways and specific transcription factors like Yap1, Met4, Msn2/4 and Pdr1/3 as the main differentiated components of the inhibitortolerant strain, which may have a role in the complex genetics of HMF and furfural tolerance [110]. ...
Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker’s yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.
... In S. cerevisiae, the toxic aldehyde group of furfural can be reduced to a hydroxyl group by multiple enzymes, including alcohol dehydrogenases (Adh1p, Adh6p, and Adh7p), methylglyoxal reductase (Ari1p), and aldehyde reductase (Gre3p), using NADH as the cofactor (36). Previous studies have linked the higher activity levels of these enzymes to enhanced furfural tolerance in multiple yeast strains (41)(42)(43). Our analysis suggests that the increased NADH-dependent furfural reduction ability and decreased ROS accumulation (Fig. 5) could, at least partially, contribute to the improved furfural tolerance in the monosomic chromosome IX mutants. ...
Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications.
... Furfural is one the most well-known lignocellulose-derived inhibitors that cause intracellular oxygen radical species accumulation. The oxidizing environment induced by ROS is known to cause irregularity and inactivity of cell components in S. cerevisiae (Almeida et al. 2008;Allen et al. 2010;Liu et al. 2020). It has been demonstrated that furfural is a thiol-reactive electrophile that can lead to oxidative stress (Kim et al. 2013;Liu et al. 2020) by activating transcription factors that mediate S. cerevisiae's response to oxidative stress (Toone et al. 1999). ...
... The oxidizing environment induced by ROS is known to cause irregularity and inactivity of cell components in S. cerevisiae (Almeida et al. 2008;Allen et al. 2010;Liu et al. 2020). It has been demonstrated that furfural is a thiol-reactive electrophile that can lead to oxidative stress (Kim et al. 2013;Liu et al. 2020) by activating transcription factors that mediate S. cerevisiae's response to oxidative stress (Toone et al. 1999). In this study, following 2 h of treatment with 35 mM furfural, 14% and 12% of the cells of the strain overexpressing YPR015C and the parental strain, respectively, exhibited a positive oxygen radical signal (Fig. 3b). ...
... Reactive oxygen species, a reduced form of superoxide anion, and hydroxyl radicals, cause high damages to the cellular components such as lipids proteins, and DNA in S. cerevisiae. The oxidative damage stress response began as a defensive system against reactive oxidants in S. cerevisiae, as in most aerobically developing organisms (Liu and Moon 2009;Liu et al. 2020). In previous studies, four genes essential for oxidative stress response to inhibitory compounds were identified in the transcriptome analysis of an industrial yeast strain (Liu and Ma 2020;Liu et al. 2020). ...
Lignocellulosic biomass is still considered a feasible source of bioethanol production. Saccharomyces cerevisiae can adapt to detoxify lignocellulose-derived inhibitors, including furfural. Tolerance of strain performance has been measured by the extent of the lag phase for cell proliferation following the furfural inhibitor challenge. The purpose of this work was to obtain a tolerant yeast strain against furfural through overexpression of YPR015C using the in vivo homologous recombination method. The physiological observation of the overexpressing yeast strain showed that it was more resistant to furfural than its parental strain. Fluorescence microscopy revealed improved enzyme reductase activity and accumulation of oxygen reactive species due to the harmful effects of furfural inhibitor in contrast to its parental strain. Comparative transcriptomic analysis revealed 79 genes potentially involved in amino acid biosynthesis, oxidative stress, cell wall response, heat shock protein, and mitochondrial-associated protein for the YPR015C overexpressing strain associated with stress responses to furfural at the late stage of lag phase growth. Both up- and down-regulated genes involved in diversified functional categories were accountable for tolerance in yeast to survive and adapt to the furfural stress in a time course study during the lag phase growth. This study enlarges our perceptions comprehensively about the physiological and molecular mechanisms implicated in the YPR015C overexpressing strain’s tolerance under furfural stress.
Construction illustration of the recombinant plasmid. a) pUG6-TEF1p-YPR015C, b) integration diagram of the recombinant plasmid pUG6-TEF1p-YPR into the chromosomal DNA of Saccharomyces cerevisiae .
... It was also identified 3 genes that had a positive correlation with ethanol production: ARO10, GCV1, and CHA1 (Fig. 2C). Besides its regulatory role in fermentation 101 , ARO10 acts in the detoxification of damaged amino acids and resistance to lignocellulosic compounds, such as HMF and furfural 102 . This gene was upregulated (log2FoldChange 1.09) upon exposure to 7 mM of pCA, with a positive correlation to ethanol production. ...
The production of ethanol from lignocellulosic sources presents increasingly difficult issues for the global biofuel scenario, leading to increased production costs of current second-generation (2G) ethanol when compared to first-generation (1G) plants. Among the setbacks encountered in industrial processes, the presence of chemical inhibitors from pre-treatment processes severely hinders the potential of yeasts in producing ethanol at peak efficiency. However, some industrial yeast strains have, either naturally or artificially, higher tolerance levels to these compounds. Such is the case of S. cerevisiae SA-1, a Brazilian fuel ethanol industrial strain that has shown high resistance to inhibitors produced by the pre-treatment of cellulosic complexes. Our study focuses on the characterization of the transcriptomic and physiological impact of an inhibitor of this type, p-coumaric acid (pCA), on this strain under chemostat cultivation via RNAseq and quantitative physiological data. It was found that strain SA-1 tend to increase ethanol yield and production rate while decreasing biomass yield when exposed to pCA, in contrast to pCA-susceptible strains, which tend to decrease their ethanol yield and fermentation efficiency when exposed to this substance. This suggests increased metabolic activity linked to mitochondrial and peroxisomal processes. The transcriptomic analysis also revealed a plethora of differentially expressed genes located in co-expressed clusters that are associated with changes in biological pathways linked to biosynthetic and energetical processes. Furthermore, it was also identified 20 genes that act as interaction hubs for these clusters, while also having association with altered pathways and changes in metabolic outputs, potentially leading to the discovery of novel targets for metabolic engineering toward a more robust industrial yeast strain.
... We also identi ed 3 genes that had a positive correlation with ethanol production: ARO10, GCV1, and CHA1 ( Figure 3C). Besides its regulatory role in fermentation 97 , ARO10 acts in the detoxi cation of damaged amino acids and resistance to lignocellulosic compounds, such as HMF and furfural 98 . We found this gene upregulated (log2FoldChange 1.09) upon exposure to 5 mM of pCA, with a positive correlation to ethanol production. ...
The production of ethanol from lignocellulosic sources presents increasingly difficult issues for the global biofuel scenario, leading to the increased production cost of current second-generation (2G) ethanol when compared to first-generation (1G) plants. Among the setbacks encountered in industrial processes, the presence of chemical inhibitors from pre-treatment processes severely hinders the potential of yeasts in producing ethanol at peak efficiency. However, some industrial yeast strains have, either naturally or artificially, higher tolerance levels to these compounds. Such is the case of SA-1, a Brazilian industrial strain that has shown high resistance to inhibitors produced by the pre-treatment of cellulosic complexes. Our study focuses on the characterization of the transcriptomic and physiological impact of an inhibitor of this type, p-Coumaric acid (pCA), on this strain under chemostat cultivation via RNAseq and HPLC data. We show that, when exposed to pCA, SA-1 yeasts tend to increase ethanol production while reducing overall biomass yield, as opposed to pCA-susceptible strains that tend to reduce their fermentation efficiency when exposed to this compound, suggesting increased metabolic activity associated with mitochondrial and peroxisomal processes. The transcriptomic analysis also revealed a plethora of differentially expressed genes located in co-expressed clusters that are associated with changes in biological pathways linked to biosynthetic and energetical processes. Furthermore, we also identified 20 genes that act as interaction hubs for these clusters, while also having association with altered pathways and changes in metabolic outputs, potentially leading to the discovery of novel targets for genetic engineering toward a more robust industrial yeast strain.
... While the genome composition largely remains the same, the adapted strain NRRL Y-50049 displayed distinct transcription and protein expression profiles from its progenitor in response to synergistic challenge of furfural and HMF (Liu et al. 2009;2019;Liu and Ma, 2020). Rewired networks of the furaldehyde-resistant NRRL Y-50049 have been characterized affecting many downstream pathways (Zhang et al. 2015). ...
... Rewired networks of the furaldehyde-resistant NRRL Y-50049 have been characterized affecting many downstream pathways (Zhang et al. 2015). Reprogrammed pathways and numerous molecular phenotypes of NRRL Y-50049 were defined to distinguish the innate stress response of its progenitor NRRL Y-12632 (Liu et al. 2009;2019;Liu and Huang 2020;Liu and Ma 2020;Liu 2011;2021). Different phenotypes were generally thought to be due to sequence mutations in terms of single nucleotide polymorphism (SNP). ...
... Such a low level of SNP in the adapted NRRL Y-50049 was not expected. As such, it does not convincingly support the comprehensive tolerant phenotypes in NRRL Y-50049 (Liu and Ma 2020;Liu 2021), nor correlate to its distinct reprogrammed pathways and rewired networks revealed in NRRL Y-50049 (Liu et al. 2009;2019;Zhang et al. 2015). ...
The industrial yeast Saccharomyces cerevisiae possesses a plastic genome enabling its adaptation to varied environment conditions. A more robust ethanologenic industrial yeast strain NRRL Y-50049 was obtained through laboratory adaptation that is resistant to 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF), a major class of toxic chemicals associated with lignocellulose-to-biofuel conversion. A significant amount of knowledge has been achieved in characterizing its tolerant phenotypes and molecular mechanisms of the resistance. Recent findings on a limited number of nonsynonymous SNP (single nucleotide polymorphism) detected in NRRL Y-50049 compared with its progenitor NRRL Y-12632 raised doubt of SNP roles in the tolerance adaptation. The genotype–phenotype relationship for yeast adaptation to the toxic chemicals is yet unclear. Here, we examine copy number variant (CNV) of the adapted strain NRRL Y-50049 to address phenotype-genotype relationships. As a background information, CNV of model strain S288C of the reference genome was also examined versus the industrial-type strain NRRL Y-12632. More than 200 CNVs, mostly duplication events, were detected in NRRL Y-12632 compared with the laboratory model strain S288C. Such enriched genetic background supports its more diversified phenotype response for the industrial yeast than the laboratory strain S288C. Comparing the two industrial strains, we found extra nine CNVs in the mitochondrial genome and 28 CNVs in the nuclear genome of NRRL Y-50049 versus its progenitor NRRL Y-12632. Continued DNA recombination event and high rate of CNV observed in NRRL Y-50049 versus its progenitor suggests that CNV is more impactful than SNP in association with phenotype-genotype relationships of yeast adaptation to the toxic chemical stress. COX1 and COB loci were defined as DNA recombination hotspots in the mitochondrial genome for the industrial yeast based on the high frequency of CNVs observed in these loci.
Key points
• COX1 and COB loci are identified as DNA recombination hotspots for the industrial yeast.
• The industrial yeast type strain NRRL Y-12632 possesses more CNVs vs the reference genome S288C.
• CNV is more important than SNP on phenotype-genotype relationships for yeast adaptation.
... The toxicity of isobutyraldehyde and other aldehydes has been a challenging issue in microbial aldehydes production and lignocellulose utilization [69]. To address this, mechanisms underlying aldehyde toxicity have been extensively studied [20,70,71] and general strategies alleviating the toxicity have been well-reviewed, including the reduction of aldehydes [72], the supply of NAD(P) H [73], the establishment of protein microcompartments [74], the engineering of protection and repair systems [75], the efficient secretion [76] and in situ separation [22]. While some tolerance mechanisms are specific to phenolic and cyclic aldehydes, tolerance engineering strategies based on these mechanisms might translate to aliphatic aldehydes including isobutyraldehyde. ...
Oxo chemicals are valuable chemicals for synthesizing a wide array of industrial and consumer products. However, producing of oxo chemicals is predominately through the chemical process called hydroformylation, which requires petroleum-sourced materials and generates abundant greenhouse gas. Current concerns on global climate change have renewed the interest in reducing greenhouse gas emissions and recycling the plentiful greenhouse gas. A carbon–neutral manner in this regard is producing oxo chemicals biotechnologically using greenhouse gas as C 1 feedstocks. Exemplifying isobutyraldehyde, this review demonstrates the significance of using greenhouse gas for oxo chemicals production. We highlight the current state and the potential of isobutyraldehyde synthesis with a special focus on the in vivo and in vitro scheme of C 1 -based biomanufacturing. Specifically, perspectives and scenarios toward carbon– and nitrogen–neutral isobutyraldehyde production are proposed. In addition, key challenges and promising approaches for enhancing isobutyraldehyde bioproduction are thoroughly discussed. This study will serve as a reference case in exploring the biotechnological potential and advancing oxo chemicals production derived from C 1 feedstocks.
... In the case of furfural, no toxic effects on S. cerevisiae growth were observed for concentrations up to 11.5 g/L (Divate et al., 2022), while a K. marxianus strain was reported to tolerate up to 3 g/L (Baptista et al., 2021b). Liu and Ma (2020) reported that the mixture of 1.9 g/L of furfural and 2.5 g/L of HMF in a synthetic medium caused growth repression on a S. cerevisiae strain until 76 h (Liu and Ma, 2020). Baptista et al., 2021b reported that the mixture of 3 g/L of HMF and 1 g/L of furfural was detoxified by K. marxianus after 48 h, allowing the exponential growth phase to start after this time (Baptista et al., 2021b). ...
... In the case of furfural, no toxic effects on S. cerevisiae growth were observed for concentrations up to 11.5 g/L (Divate et al., 2022), while a K. marxianus strain was reported to tolerate up to 3 g/L (Baptista et al., 2021b). Liu and Ma (2020) reported that the mixture of 1.9 g/L of furfural and 2.5 g/L of HMF in a synthetic medium caused growth repression on a S. cerevisiae strain until 76 h (Liu and Ma, 2020). Baptista et al., 2021b reported that the mixture of 3 g/L of HMF and 1 g/L of furfural was detoxified by K. marxianus after 48 h, allowing the exponential growth phase to start after this time (Baptista et al., 2021b). ...
The non-conventional yeast Kluyveromyces marxianus is widely used for several biotechnological applications, mainly due to its thermotolerance, high growth rate, and ability to metabolise a wide range of sugars. These cell traits are strategic for lignocellulosic biomass valorisation and strain diversity prompts the development of robust chassis, either with improved tolerance to lignocellulosic inhibitors or ethanol. This review summarises bioethanol and value-added chemicals production by K. marxianus from different lignocellulosic biomasses. Moreover, metabolic engineering and process optimization strategies developed to expand K. marxianus potential are also compiled, as well as studies reporting cell mechanisms to cope with lignocellulosic-derived inhibitors. The main lignocellulosic-based products are bioethanol, representing 71% of the reports, and xylitol, representing 17% of the reports. K. marxianus also proved to be a good chassis for lactic acid and volatile compounds production from lignocellulosic biomass, although the literature on this matter is still scarce. The increasing advances in genome editing tools and process optimization strategies will widen the K. marxianus-based portfolio products.
... The transcriptional regulation of GST genes also proved to be quite ambiguous under our experimental conditions (Table 4). It was expected that inhibitory PAC components might induce the increased expression of these enzymes, as this has recently been observed in S. cerevisiae in response to the addition of furfural and HMF [94]. Indeed, we were able to identify two GST genes (AO090003000631 and AO090103000485) that were upregulated on PAC compared to glucose and pure acetate. ...
Due to its acetate content, the pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw could provide an inexpensive substrate for microbial fermentation. However, PAC also contains several inhibitors that make its detoxification inevitable. In our study, we examined the transcriptional response of Aspergillus oryzae to cultivation on 20% detoxified PAC, pure acetate and glucose using RNA-seq analysis. Functional enrichment analysis of 3463 significantly differentially expressed (log2FC > 2 & FDR < 0.05) genes revealed similar metabolic tendencies for both acetate and PAC, as upregulated genes in these cultures were mainly associated with ribosomes and RNA processing, whereas transmembrane transport was downregulated. Unsurprisingly, metabolic pathway analysis revealed that glycolysis/gluconeogenesis and starch and sucrose metabolism were upregulated for glucose, whereas glyoxylate and the tricarboxylic acid (TCA) cycle were important carbon utilization pathways for acetate and PAC, respectively. Moreover, genes involved in the biosynthesis of various amino acids such as arginine, serine, cysteine and tryptophan showed higher expression in the acetate-containing cultures. Direct comparison of the transcriptome profiles of acetate and PAC revealed that pyruvate metabolism was the only significantly different metabolic pathway and was overexpressed in the PAC cultures. Upregulated genes included those for methylglyoxal degradation and alcohol dehydrogenases, which thus represent potential targets for the further improvement of fungal PAC tolerance.
