Acetic Acid Tolerance Research Articles

Differences in microbial communities among different types of zaopei and their effects on quality and flavor of baijiu

Three types of zaopei (fermented grain) of xiaoqu light-flavor baijiu (XQZP), daqu light-flavor baijiu (DQZP), and strong-flavor baijiu (SFZP) at the end of fermentation and their dominant lactic acid bacteria were systematically compared and analyzed in this study. The results showed that these three types of zaopei differed significantly in acidity, reducing sugar content, and ethanol content, and that the main factors influencing their microbial community were acidity and lactic acid. The diversity and contents of flavor substances were significantly higher in SFZP than in DQZP and XQZP. Additionally, there was a strong correlation between dominant lactic acid bacteria and flavor substances in all three zaopei, but the correlation between fungi and flavor substances was higher than that between bacteria and flavor substances. Differential gene analysis revealed that the microbial activities followed the order of SFZP > DQZP > XQZP. The KEGG enrichment analysis indicated that the differential genes from different zaopei were enriched in different metabolic pathways. Furthermore, various microorganisms in 3 types of zaopei contained different functional genes, of which fungi mainly contained genes responsible for the synthesis of ethanol and acetic acid, while lactic acid bacteria mainly contained genes responsible for the synthesis of lactic acid. In XQZP, L. helveticus was dominant lactic acid bacteria prominent in acetic acid tolerance and lactic acid production; in DQZP, L. acetotolerans was remarkable in its tolerance to lactic acid, acetic acid, ethanol and lactic acid production; and in SFZP, A. jinshanensis was superior in acetic acid tolerance and production. Taken together, this study reveals the mechanism underlying flavor differences among three types of baijiu and provides valuable references for the development and utilization of baijiu microbial resources.

Improving the alcohol respiratory chain and energy metabolism by enhancing PQQ synthesis in Acetobacter pasteurianus.

Pyrroquinoline quinone (PQQ) is one of the important coenzymes in living organisms. In acetic acid bacteria (AAB) it plays a crucial role in alcohol respiratory chain, as a coenzyme of alcohol dehydrogenase. In this work, the PQQ biosynthetic genes were overexpressed in Acetobacter pasteurianus CGMCC 3089 to improve the fermentation performance. The result shows that the intracellular and extracellular PQQ contents in the recombinant strain A. pasteurianus (pBBR1-p264-pqq) were 152.53% and 141.08% higher than those of the control A. pasteurianus (pBBR1-p264), respectively. The catalytic activity of alcohol dehydrogenase and aldehyde dehydrogenase increased by 52.92% and 67.04%, respectively. The results indicated that the energy charge and intracellular ATP were also improved in the recombinant strain. The acetic acid fermentation was carried out using a 5 L self-aspirating fermenter, and the acetic acid production rate of the recombinant strain was 23.20% higher compared with the control. Furthermore, the relationship between the PQQ and acetic acid tolerance of cells was analyzed. The biomass of recombinant strain was 180.2%, 44.3%, and 38.6% higher than those of control under 2%, 3%, and 4% acetic acid stress, respectively. After treated with 6% acetic acid for 40 min, the survival rate of the recombinant strain was increased by 76.20% compared with the control. Those result demonstrated that overexpression of PQQ biosynthetic genes increased the content of PQQ, therefore improving the acetic acid fermentation and the cell tolerance against acetic acid by improving the alcohol respiratory chain and energy metabolism.

Overexpression of arginase gene CAR1 renders yeast Saccharomyces cerevisiae acetic acid tolerance

Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1, renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1-overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol.

The role of ion homeostasis in adaptation and tolerance to acetic acid stress in yeasts.

Maintenance of asymmetric ion concentrations across cellular membranes is crucial for proper yeast cellular function. Disruptions of these ionic gradients can significantly impact membrane electrochemical potential and the balance of other ions, particularly under stressful conditions such as exposure to acetic acid. This weak acid, ubiquitous to both yeast metabolism and industrial processes, is a major inhibitor of yeast cell growth in industrial settings and a key determinant of host colonization by pathogenic yeast. Acetic acid toxicity depends on medium composition, especially on the pH (H+ concentration), but also on other ions' concentrations. Regulation of ion fluxes is essential for effective yeast response and adaptation to acetic acid stress. However, the intricate interplay among ion balancing systems and stress response mechanisms still presents significant knowledge gaps. This review offers a comprehensive overview of the mechanisms governing ion homeostasis, including H+, K+, Zn2+, Fe2+/3+, and acetate, in the context of acetic acid toxicity, adaptation, and tolerance. While focus is given on Saccharomyces cerevisiae due to its extensive physiological characterization, insights are also provided for biotechnologically and clinically relevant yeast species whenever available.

Acetic acid- and furfural-based adaptive evolution of Saccharomyces cerevisiae strains for improving stress tolerance and lignocellulosic ethanol production

Acetic acid and furfural are known as prevalent inhibitors deriving from pretreatment during lignocellulosic ethanol production. They negatively impact cell growth, glucose uptake and ethanol biosynthesis of Saccharomyces cerevisiae strains. Development of industrial S. cerevisiae strains with high tolerance towards these inhibitors is thus critical for efficient lignocellulosic ethanol production. In this study, the acetic acid or furfural tolerance of different S. cerevisiae strains could be significantly enhanced after adaptive evolution via serial cultivation for 40 generations under stress conditions. The acetic acid-based adaptive strain SPSC01-TA9 produced 30.5 g·L–1 ethanol with a yield of 0.46 g·g–1 in the presence of 9 g·L–1 acetic acid, while the acetic acid/furfural-based adaptive strain SPSC01-TAF94 produced more ethanol of 36.2 g·L–1 with increased yield up to 0. 49 g·g–1 in the presence of both 9 g·L–1 acetic acid and 4 g·L–1 furfural. Significant improvements were also observed during non-detoxified corn stover hydrolysate culture by SPSC01-TAF94, which achieved ethanol production and yield of 29.1 g·L–1 and 0.49 g·g–1, respectively, the growth and fermentation efficiency of acetic acid/furfural-based adaptive strain in hydrolysate was 95% higher than those of wildtype strains, indicating the acetic acid- and furfural-based adaptive evolution strategy could be an effective approach for improving lignocellulosic ethanol production. The adapted strains developed in this study with enhanced tolerance against acetic acid and furfural could be potentially contribute to economically feasible and sustainable lignocellulosic biorefinery.

Novel Roles of the Greatwall Kinase Rim15 in Yeast Oxidative Stress Tolerance through Mediating Antioxidant Systems and Transcriptional Regulation.

The Greatwall-family protein kinase Rim15 is associated with the nutrient starvation response, whereas its role in oxidative stress responses remains unclear. Here, acetic acid and peroxide were used as two oxidative stress elicitors. The antioxidant indicator assay under acetic acid stress revealed the impaired growth in rim15Δ related to the regulation of antioxidant systems. Comparative transcriptome analysis revealed that differentially expressed genes (DEGs) are predicted to be mostly regulated by oxidative stress-responsive transcriptional factor Yap1. Among the DEGs, acetic acid stress-induced genes were found, and YAP1 disruption also inhibited their induction. The deletion of Rim15 or the Rim15 kinase domain in yap1Δ did not further decrease the gene expression, suggesting that Rim15 functions together with Yap1 in regulating acetic acid stress-induced genes, which requires Rim15 kinase activity. Additionally, Rim15 regulated H2O2 stress tolerance through partially similar but special mechanisms in that Rim15 kinase activity impacted acetic acid and H2O2 stress tolerance in different degrees, indicating the different mechanisms underlying Rim15-mediated redox regulation against different stressors. These results benefit the better understanding of stress signaling pathways related to Rim15. Given that Rim15 and some of its target genes are conserved across eukaryotes, these results also provide a basis for studies of oxidative stress-related processes in other organisms.

Genome-scale transcriptional activation by non-homologous end joining-mediated integration in Yarrowia lipolytica

BackgroundGenome-scale screening can be applied to efficiently mine for unknown genes with phenotypes of interest or special functions. It is also useful to identify new targets for engineering desirable properties of cell factories.ResultsHere, we designed a new approach for genome-scale transcription activation using non-homologous end joining (NHEJ)-mediated integration in Yarrowia lipolytica. We utilized this approach to screen for genes that, upon activation, confer phenotypes including improved acetic acid tolerance and xylose metabolism. The candidates were validated using gene overexpression, and functional changes including improved growth performance under multiple stressors and activated pentose metabolism were identified.ConclusionsThis study provides a simple and effective approach to randomly activate endogenous genes and mine for key targets associated with phenotypes of interest. The specific gene targets identified here will be useful for cell factory construction and biorefining lignocellulose.

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0–6.4% decrease), glycerol (31.4–78.5% increase), and acetic acid (53.0–60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications.Key points•Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii•Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid•Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genesGraphical

The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis.

Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic performance of Saccharomyces cerevisiae. The adaptive response and tolerance to this stress involves the activation of a complex network of molecular pathways. This study aims to delve deeper into these mechanisms in S. cerevisiae, particularly focusing on the role of the Hrk1 kinase. Hrk1 is a key determinant of acetic acid tolerance, belonging to the NPR/Hal family, whose members are implicated in the modulation of the activity of plasma membrane transporters that orchestrate nutrient uptake and ion homeostasis. The influence of Hrk1 on S. cerevisiae adaptation to acetic acid-induced stress was explored by employing a physiological approach based on previous phosphoproteomics analyses. The results from this study reflect the multifunctional roles of Hrk1 in maintaining proton and potassium homeostasis during different phases of acetic acid-stressed cultivation. Hrk1 is shown to play a role in the activation of plasma membrane H+-ATPase, maintaining pH homeostasis, and in the modulation of plasma membrane potential under acetic acid stressed cultivation. Potassium (K+) supplementation of the growth medium, particularly when provided at limiting concentrations, led to a notable improvement in acetic acid stress tolerance of the hrk1Δ strain. Moreover, abrogation of this kinase expression is shown to confer a physiological advantage to growth under K+ limitation also in the absence of acetic acid stress. The involvement of the alkali metal cation/H+ exchanger Nha1, another proposed molecular target of Hrk1, in improving yeast growth under K+ limitation or acetic acid stress, is proposed.

Integrated fermentative process for lipid and β-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast

An engineered Yarrowia lipolytica strain was successfully employed to produce β-carotene and lipids from acetic acid, a product of syngas fermentation by Clostridium aceticum. The strain showed acetic acid tolerance up to concentrations of 20 g/L. Flask experiments yielded a peak lipid content of 33.7 % and β-carotene concentration of 13.6 mg/g under specific nutrient conditions. The study also investigated pH effects on production in bioreactors, revealing optimal lipid and β-carotene contents at pH 6.0, reaching 22.9 % and 44 mg/g, respectively. Lipid profiles were consistent across experiments, with C18:1 being the dominant compound at approximately 50 %. This research underscores a green revolution in bioprocessing, showing how biocatalysts can convert syngas, a potentially polluting byproduct, into valuable β-carotene and lipids with a Y. lipolytica strain.

Adaptive laboratory evolution for acetic acid-tolerance matches sourdough challenges with yeast phenotypes

Acetic acid tolerance of Saccharomyces cerevisiae is an important trait in sourdough fermentation processes, where the accumulation of acid by the growth of lactic acid bacteria reduces the yeast metabolic activity. In this work, we have carried out adaptive laboratory evolution (ALE) experiments in two sourdough isolates of S. cerevisiae exposed to acetic acid, or alternatively to acetic acid and myriocin, an inhibitor of sphingolipid biosynthesis that sped-up the evolutionary adaptation. Evolution approaches resulted in acetic tolerance, and surprisingly, increased lactic susceptibility. Four evolved clones, one from each parental strain and evolutionary scheme, were selected on the basis of their potential for CO2 production in sourdough conditions. Among them, two showed phenotypic instability characterized by strong lactic sensitivity after several rounds of growth under unstressed conditions, while two others, displayed increased constitutive acetic tolerance with no loss of growth in lactic medium. Genome sequencing and ploidy level analysis of all strains revealed aneuploidies, which could account for phenotypic heterogeneity. In addition, copy number variations (CNVs), affecting specially to genes involved in ion transport or flocculation, and single nucleotide polymorphisms (SNPs) were identified. Mutations in several genes, ARG82, KEX1, CTK1, SPT20, IRA2, ASG1 or GIS4, were confirmed as involved in acetic and/or lactic tolerance, and new determinants of these phenotypes, MSN5 and PSP2, identified.

The facilitation between Staphylococcus carnosus M43 and Zygosaccharomyces rouxii Y-8, and as starter on the quality of broad bean paste

Zygosaccharomyces rouxii (Z. rouxii) and Staphylococcus carnosus (S. carnosus) are fundamental taxa in spontaneous bean-based food fermentation, playing a vital role in flavor generation and macromolecule degradation. The present study aimed to investigate the biological mechanisms underlying the interaction between Z. rouxii Y-8 and S. carnosus M43 in BBP (broad bean paste) moromi by mono- and co-culture. It was found that the biomass of Z. rouxii and S. carnosus was higher in co-culture than that in mono-culture (p < 0.05), indicating that a facilitation relationship among them. Moreover, the observed intensity of facilitation (OIF) peaked at 0.479 ± 0.002 under aerobic conditions, which was higher than that under anaerobic conditions (0.147 ± 0.014). Furthermore, the facilitative microbial interaction showed the stability and robustness during co-culture with different inoculum proportions (1:3, 1:5, 1:7, 1:10) and cell passage from 1 to 4 generations. Physical separation co-culture and acetic acid tolerance test revealed that acetic acid played a crucial role in regulating the interaction between Z. rouxii (R2 = 0.6955) and S. carnosus (R2 = 0.7097), with metabolic complementarity and inhibition alleviation as the underlying mechanisms. Additionally, Z. rouxii and S. carnosus as starter could enhance the accumulation of flavor compound, and accelerate the maturity of BBP fermentation. The dissection of interaction between Z. rouxii and S. carnosus provided valuable insights for revealing the complex web of relationships among microbiota and the development of microbial starter in BBP brewing ecosystem.

Isolation, Identification, and Characterization of an Acid-Tolerant Pichia kudriavzevii and Exploration of Its Acetic Acid Tolerance Mechanism

Pichia kudriavzevii is one of the major non-Saccharomyces cerevisiae yeasts in Chinese baijiu brewing, which has shown a substantially higher tolerance to acid, heat, and ethanol. Exploring the mechanism of P. kudriavzevii could have a positive effect on the artificially controlled production of baijiu. In this study, an efficient acetic-acid-tolerant P. kudriavzevii strain, Y2, was isolated from the yellow water of strong-flavored baijiu brewing waste, and its molecular mechanism of acetic acid tolerance was investigated through a comparative transcriptomic analysis. The strain Y2 could tolerate 12 g/L of acetic acid. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, differentially expressed genes (DEGs) were mainly enriched in oxidative phosphorylation, the citrate cycle, glycolysis/gluconeogenesis, and carbon metabolism under low (AL group) and high (AH group) concentrations of acetic acid. However, the DEG enrichment was more profound in the AH group when compared to the control. Compared with the AL group, the expression of genes related to oxidative phosphorylation was more significantly upregulated, while in terms of the TCA cycle, phosphoenolpyruvate carboxykinase was significantly upregulated in both the AH and AL groups and was positively correlated with tolerance to acetic acid. This was followed by citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase. These results illustrated a possible mechanism of acid tolerance by regulating the metabolism-related pathways in P. kudriavzevii and provided a basis for the further investigation of the acid tolerance mechanism.

Effect and application of proline metabolism-related gene CgMCUR1 on stress tolerance of Candida glycerinogenes and Saccharomyces cerevisiae.

To investigate the effect of CgMCUR1 on the phenotype of Candida glycerinogenes and Saccharomyces cerevisiae. Inhibition of CgMCUR1 expression reduced acetate, H2O2, and high temperature tolerance of C. glycerinogenes. Expression of CgMCUR1 resulted in better acetic acid, H2O2, and high temperature tolerance in recombinant S. cerevisiae. Meanwhile, CgMCUR1 was able to enhance intracellular proline accumulation. The qRT-PCR analysis revealed that overexpression of CgMCUR1 affected proline metabolism in recombinant S. cerevisiae. The overexpression strain also showed reduced levels of cellular lipid peroxidation and an altered ratio of saturated fatty acid (SFA) to unsaturated fatty acid (UFA) in the cell membrane. The ethanol production of recombinant S. cerevisiae at high temperature was 30.9g l-1, obtaining an increase of 12%, and the conversion rate was increased by 12%. In the undetoxified cellulose hydrolysate, the ethanol yield was 14.7g l-1 at 30h with an improvement of 18.5%, and the conversion rate was increased by 15.3%. Overexpression of CgMCUR1 rendered the acetic acid, H2O2, and high temperature tolerant of recombinant S. cerevisiae and enhanced the ethanol fermentation performance of recombinant S. cerevisiae under high temperature stress and in undetoxified cellulose hydrolysate by improving intracellular proline accumulation and by changing cellular physiological metabolism.

Genomic Plasticity of Acid-Tolerant Phenotypic Evolution in Acetobacter pasteurianus.

Acetic acid bacteria have a remarkable capacity to cope with elevated concentrations of cytotoxic acetic acid in their fermentation environment. In particular, the high-level acetate tolerance of Acetobacter pasteurianus that occurs in vinegar industrial settings must be constantly selected for. However, the improved acetic acid tolerance is rapidly lost without a selection pressure. To understand genetic and molecular biology of this acquired acetic acid tolerance in A. pasteurianus, we evolved three strains A. pasteurianus CICIM B7003, CICIM B7003-02, and ATCC 33,445 over 960 generations (4months) in two initial acetic acids of 20g·L-1 and 30g·L-1, respectively. An acetic acid-adapted strain M20 with significantly improved specific growth rate of 0.159h-1 and acid productivity of 1.61g·L-1·h-1 was obtained. Comparative genome analysis of six evolved strains revealed that the genetic variations of adaptation were mainly focused on lactate metabolism, membrane proteins, transcriptional regulators, transposases, replication, and repair system. Among of these, lactate dehydrogenase, acetolactate synthase, glycosyltransferase, ABC transporter ATP-binding protein, two-component regulatory systems, the type II toxin-antitoxin system (RelE/RelB/StbE), exodeoxyribonuclease III, type I restriction endonuclease, tRNA-uridine 2-sulfurtransferase, and transposase might collaboratively contribute to the improved acetic acid tolerance in A. pasteurianus strains. The balance between repair factors and transposition variations might be the basis for genomic plasticity of A. pasteurianus strains, allowing the survival of populations and their offspring in acetic acid stress fluctuations. These observations provide important insights into the nature of acquired acetic acid tolerance phenotype and lay a foundation for future genetic manipulation of these strains.

CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae

Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.

New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution

The production of yeast oil from lignocellulosic biomasses is impaired by inhibitors formed during the pretreatment step, mainly acetic acid. Herein, we applied Adaptive Laboratory Evolution (ALE) to select three Acetic acid Tolerant Strains (ATS) of P. laurentii UFV-1. Different phenotypes emerged alongside evolution. The ATS II presented trade-offs in the absence of acetic acid, suggesting that it displays a specialized phenotype of tolerance to growth on organic acids. On the other hand, ATS I and ATS III presented phenotypes associated with the behavior of generalists. ATS I was considered the most promising evolved strain as it displayed the oleaginous phenotype in all conditions tested. Thus, we applied whole-genome sequencing to detect the mutations that emerged in this strain during the ALE. We found alterations in genes encoding proteins involved in different cellular functions, including multidrug resistance (MDR) transporters, energy metabolism, detoxification, coenzyme recycling, and cell envelope remodeling. To evaluate acetic acid stress responses, both parental and ATS I strains were cultivated in chemostat mode in the absence and presence of acetic acid. In contrast to ATS I, the parental strain presented alterations in the cell envelope and cell size under acetic acid stress conditions. Furthermore, the parental strain and the ATS I presented differences regarding acetic acid assimilation. Contrary to the parental strain, the ATS I displayed an increase in unsaturated fatty acid content irrespective of acetic acid stress, which might be related to improved tolerance to acetic acid. Altogether, these results provided insights into the mechanisms involved with the acetic acid tolerance displayed by ATS I and the responses of P. laurentii to this stressful condition.

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

BackgroundAcetic 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.ResultsAn 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.ConclusionsThis 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.

Exploring Natural Fermented Foods as a Source for New Efficient Thermotolerant Yeasts for the Production of Second-Generation Bioethanol

Considering the cost-effectiveness of bioethanol production at high temperatures, there is an enduring need to find new thermotolerant ethanologenic yeasts. In this study, a total of eighteen thermotolerant yeasts were isolated from various natural fermented products in Morocco. Ethanol production using 50 g/L glucose or 50 g/L xylose as the sole carbon source revealed potential yeasts with high productivities and volumetric ethanol productivities at high temperatures. Based on molecular identification, the selected thermotolerant fermentative isolates were affiliated with Pichia kudriavzevii, Kluyveromyces marxianus, and Kluyveromyces sp. During the simultaneous saccharification and fermentation of lignocellulosic biomass at a high temperature (42 °C), the designated yeast P. kudriavzevii YSR7 produced an ethanol concentration of 22.36 g/L, 18.2 g/L and 6.34 g/L from 100 g/L barley straw (BS), chickpea straw (CS), and olive tree pruning (OTP), respectively. It also exhibited multi-stress tolerance, such as ethanol, acetic acid, and osmotic tolerance. Therefore, the yeast P. kudriavzevii YSR7 showed promising attributes for biorefinery-scale ethanol production in the future.

Combined roles of exporters in acetic acid tolerance in Saccharomyces cerevisiae

Acetic acid is a growth inhibitor generated during alcoholic fermentation and pretreatment of lignocellulosic biomass, a major feedstock to produce bioethanol. An understanding of the acetic acid tolerance mechanisms is pivotal for the industrial production of bioethanol. One of the mechanisms for acetic acid tolerance is transporter-mediated secretion where individual transporters have been implicated. Here, we deleted the transporters Aqr1, Tpo2, and Tpo3, in various combinations, to investigate their combined role in acetic acid tolerance. Single transporter deletions did not impact the tolerance at mild acetic acid stress (20 mM), but at severe stress (50 mM) growth was decreased or impaired. Tpo2 plays a crucial role in acetic acid tolerance, while the AQR1 deletion has a least effect on growth and acetate efflux. Deletion of both Tpo2 and Tpo3 enhanced the severe growth defects at 20 mM acetic acid concomitantly with a reduced rate of acetate secretion, while TPO2 and/or TPO3 overexpression in ∆tpo2∆tpo3∆ restored the tolerance. In the deletion strains, the acetate derived from sugar metabolism accumulated intracellularly, while gene transcription analysis suggests that under these conditions, ethanol metabolism is activated while acetic acid production is reduced. The data demonstrate that Tpo2 and Tpo3 together fulfill an important role in acetate efflux and the acetic acid response.