Acetic Acid Stress Research Articles

Acetic acid stress in budding yeast: From molecular mechanisms to applications.

Acetic acid stress represents a frequent challenge to counteract for yeast cells under several environmental conditions and industrial bioprocesses. The molecular mechanisms underlying its response have been mostly elucidated in the budding yeast Saccharomyces cerevisiae, where acetic acid can be either a physiological substrate or a stressor. This review will focus on acetic acid stress and its response in the context of cellular transport, pH homeostasis, metabolism and stress‐signalling pathways. This information has been integrated with the results obtained by multi‐omics, synthetic biology and metabolic engineering approaches aimed to identify major cellular players involved in acetic acid tolerance. In the production of biofuels and renewable chemicals from lignocellulosic biomass, the improvement of acetic acid tolerance is a key factor. In this view, how this knowledge could be used to contribute to the development and competitiveness of yeast cell factories for sustainable applications will be also discussed.

Functional metaproteomic analysis of alcohol vinegar microbiota during an acetification process: A quantitative proteomic approach

Vinegar is elaborated using a semi-continuous submerged culture of a complex microbiota of acetic acid bacteria. The genus Komagataeibacter provides much of the proteins of the metaproteome, being K. europaeus the main species working in this environment. In this work, the protein profile of the vinegar microbiota, obtained by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples from different cycle times of an acetification process using an alcohol medium, has been used to describe the functional metaproteome throughout the process. The analysis was focused on Komagataeibacter species which supplied about 90% of the metaproteome and particularly K. europaeus which accounts for more than 70%. According to these results, the natural behaviour of a microbial community in vinegar has been predicted at a quantitative proteomic level. The results revealed that most of the identified proteins involved in the metabolism of amino acids, biosynthesis of proteins, and energy production related-metabolic pathways increased their expression throughout the cycle loading phase and afterwards experimented a decrease coming into play other proteins acting against acetic acid stress. These findings may facilitate a better understanding of the microbiota's role and contributing to obtain a quality product.

Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

BackgroundSaccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown.ResultsIn this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance.ConclusionTranslation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

Effect of high pressure processing, allyl isothiocyanate, and acetic acid stresses on Salmonella survivals, storage, and appearance color in raw ground chicken meat

High pressure processing (HPP) technique combined with food-grade antimicrobials, i.e. allyl isothiocyanate (AITC, a major compound in mustard oil) and acetic acid (AA, a key component in vinegar), is a promising preservative hurdle system for meat industries to reduce foodborne pathogens. With the addition of AITC and AA, the hydrostatic pressure level may be lowered to 250–350 MPa which clearly demonstrated a synergistic effect on the reduction of Salmonella. For example, a 5 log reduction was obtained at 350 MPa (4 min) combined with 0.05% AITC (w/w); and more than 7 log inactivation was achieved at 350 MPa (12 min) with 0.075% AITC and 0.1% AA (w/w) added. The color of ground chicken meat is mainly affected by pressurization, where color coordinates (L*, a*, b*), and ΔE were significantly increased after treated with 250 MPa for 4 min (P < 0.05). In addition, the storage test (at 4 °C and 10 °C) after HPP (300 MPa, 8 min)+AITC (0.0625%)+AA (0.05%) treatment further confirmed that AITC and AA potentially remained active and continuously killed the stress-damaged cells up to 28 days. Linear regression model (R2 = 0.94) and dimensionless non-linear model [F-value = 910.07; Pr > F (<0.0001)] were developed and validated to predict the impact of the four factors, i.e., pressure level (250–350 MPa), operation time (4–12 min), and concentrations of AITC (0.05–0.075%) and AA (0.0–0.1%), on Salmonella survival in ground chicken meat and all discrepancies (prediction vs. experiment) were within 15%. Those models may provide useful tools for regulatory agencies on the microbial risk assessment to enhance food safety and for food industries on process optimization and cost reduction.

Transcriptome response of Acetobacter pasteurianus Ab3 to high acetic acid stress during vinegar production.

Acetic acid accumulation is a universal limiting factor to the vinegar manufacture because of the toxic effect of acetic acid on the acid producing strain, such as Acetobacter pasteurianus. In this study, we aimed to investigate the genome-wide transcriptional response of A. pasteurianus Ab3 to high acid stress during vinegar production. By comparing the transcriptional landscape of cells harvested from a long-term cultivation with high acidity (70 ± 3g/L) to that of low acidity (10 ± 2g/L), we demonstrated that 1005 genes were differentially expressed. By functional enrichment analysis, we found that the expression of genes related to the two-component systems (TCS) and toxin-antitoxin systems (TAS) was significantly regulated under high acid stress. Cells increased the genome stability to withstand the intracellular toxicity caused by the acetic acid accumulation by repressing the expression of transposases and integrases. Moreover, high acid stress induced the expression of genes involved in the pathways of peptidoglycan, ceramide, and phosphatidylcholine biosynthesis as well as the Tol-Pal and TonB-ExbB systems. In addition, we observed that cells increased and diversified the ATP production to resist high acid stress. Transcriptional upregulation in the pathways of pyrroloquinoline quinone (PQQ) synthesis and thiamine metabolism suggested that cells may increase the production of prosthetic groups to ensure the enzyme activity upon high acid stress. Collectively, the results of this study increase our current understanding of the acetic acid resistance (AAR) mechanisms in A. pasteurianus and provide opportunities for strain improvement and scaled-up vinegar production.Key Points• TCS and TAS are responsive to the acid stress and constitute the regulating networks.• Adaptive expression changes of cell envelope elements help cell resist acid stress.• Cells promote genome stability and diversify ATP production to withstand acid stress.

Impact of hfq and sigE on the tolerance of Zymomonas mobilis ZM4 to furfural and acetic acid stresses.

Zymomonas mobilis, as an ethanologenic microorganism with many desirable industrial features, faces crucial obstacles in the lignocellulosic ethanol production process. A significant hindrance occurs during the pretreatment procedure that not only produces fermentable sugars but also releases severe toxic compounds. As diverse parts of regulation networks are involved in different aspects of complicated tolerance to inhibitors, we developed ZM4-hfq and ZM4-sigE strains, in which hfq and sigE genes were overexpressed, respectively. ZM4-hfq is a transcription regulator and ZM4-sigE is a transcription factor that are involved in multiple stress responses. In the present work, by overexpressing these two genes, we evaluated their impact on the Z. mobilis tolerance to furfural, acetic acid, and sugarcane bagasse hydrolysates. Both recombinant strains showed increased growth rates and ethanol production levels compared to the parental strain. Under a high concentration of furfural, the growth rate of ZM4-hfq was more inhibited compared to ZM4-sigE. More precisely, fermentation performance of ZM4-hfq revealed that the yield of ethanol production was less than that of ZM4-sigE, because more unused sugar had remained in the medium. In the case of acetic acid, ZM4-sigE was the superior strain and produced four and two-fold more ethanol compared to the parental strain and ZM4-hfq, respectively. Comparison of inhibitor tolerance between single and multiple toxic inhibitors in the fermentation of sugarcane bagasse hydrolysate by ZM4-sigE strain showed similar results. In addition, ethanol production performance was considerably higher in ZM4-sigE as well. Finally, the results of the qPCR analysis suggested that under both furfural and acetic acid treatment experiments, overproduction of both hfq and sigE improves the Z. mobilis tolerance and its ethanol production capability. Overall, our study showed the vital role of the regulatory elements to overcome the obstacles in lignocellulosic biomass-derived ethanol and provide a platform for further improvement by directed evolution or systems metabolic engineering tools.

Investigation of lipid profile in Acetobacter pasteurianus Ab3 against acetic acid stress during vinegar production.

Elucidation of the acetic acid resistance (AAR) mechanisms of Acetobacter pasteurianus is significant for vinegar production. In this study, cell membrane lipid profile of A. pasteurianus Ab3 was investigated by gas chromatography-mass spectrometer (GC-MS) and high performance liquid chromatography-electrospray ionization (HPLC-ESI) combined with high resolution accurate mass/mass spectrometry (HRAM/MS). We observed that cell remodeled the membrane physical state by decreasing the ratio of saturated fatty acids (SFAs)/unsaturated fatty acids (UFAs), and increasing the chain length of fatty acids (FAs) and the content of cyclopropane FAs in response to extreme acid stress. Noticeably, the content of octadecadienoic acid (C18:2) elevated remarkably. Moreover, a continuous reduction in cell membrane fluidity and a "V-type" variance in permeability were discovered. The content of glycerophospholipid and ceramide increased significantly in cells harvested from culture with acidity of 75g/L and 95g/L compared to that with acidity of 30g/L. Among the identified lipid species, the content of phosphatidylcholine (e.g. PC 19:0/18:2 and 19:1/18:0), ceramide (e.g. Cer d18:0/16:1 and d18:0/16:1 + O), and dimethylphosphatidylethanolamine (e.g. dMePE 19:1/16:1) increased notably with increasing acidity. Collectively, these findings refresh our current understanding of the AAR mechanisms in A. pasteurianus Ab3, and should direct future strain breeding and vinegar fermentation.

CADM1 enhances intestinal barrier function in a rat model of mild inflammatory bowel disease by inhibiting the STAT3 signaling pathway.

Cell adhesion molecule 1 (CADM1) is frequently silenced in lung, prostate, liver, stomach, pancreatic and breast carcinomas and other forms of human carcinomas. However, it is unclear regarding the role of CADM1 in irritable bowel syndrome with diarrhoea (IBS-D) that is the most common gastrointestinal diagnosis and may contribute to impaired intestinal barrier function. The aim of the present study is to explore the potential mechanism of CADM1 in regulating intestinal barrier function in IBS-D. A rat model with IBS-D induced by the combination method of mother-infant separation, acetic acid and restraint stress was initially established. The defecation frequency, faecal water content (FWC), total intestinal permeability, sIgA, endotoxin, D-lactic acid and diamine oxidase (DAO) were then measured. Next, positive expression of CADM1 protein was detected in distal colonic tissue of rats by immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in distal colonic mucosa, CADM1, Janus kinase 1 (JAK1), STAT3, p-JAK1, p-STAT3, Claudin-1and Claudin-2 were evaluated using ELISA, RT-qPCR and western blot analysis. IBS-D rats exhibited low CADM1 expression and activated STAT3 signaling pathway. Overexpression of CADM1 in rats was shown to increase Claudin-1 expression, while decreasing expression of STAT3, Claudin-2, TNF-α and IL-6. In addition, silencing of CADM1 or inhibition of the STAT3 signaling pathway was demonstrated to improve the intestinal barrier function. Our study provides evidence that CADM1 can potentially improve intestinal barrier function in rats with IBS-D by inhibiting the STAT3 signaling pathway.

Integrated transcriptomic and proteomic analysis of the acetic acid stress in Issatchenkia orientalis.

Issatchenkia orientalis known as a multi-tolerant non-Saccharomyces yeast, which tolerant environmental stresses, exhibits potential in wine making and bioethanol production. It is essential for the growth of I.orientalis to tolerant acetic acid in the mixed cultures with Saccharomyces cerevisiae. In this work, RNA-sequence and TMT (Tandem Mass Tag) were used to examine the comprehensive transcriptomic and proteomic profiles of I.orientalis in response to acetic acid. The results showed that 876 genes were identified differentially transcribed in I.orientalis genome and 399 proteins expressed in proteome after 4hr acetic acid (90mM, pH 4.5). The comprehensive analysis showed a series of determinants of acetic acid tolerance: Glycolysis and TCA cycle provide enough nicotinamide adenine dinucleotide to effectively convert acetic acid. Genes associated with potassium, iron, zinc, and glutathione synthesis were upregulated. The same changes of differentially expressed genes and proteins were mainly concentrated in chaperones, coenzyme, energy production, and transformation. PRACTICAL APPLICATIONS: In addition to the main fermentation products, wine yeast also produces metabolite acetic acid in the fermentation process, and yeast cells are exposed to acetic acid stress, which restrains cell proliferation. Issatchenkia orientalis exhibits great potential in winemaking and bioethanol production. The yeast is known as a multi-tolerant non-Saccharomyces yeast that can tolerate a variety of environmental stresses. In this study, RNA-Seq and TMT were conducted to investigate the changes in transcriptional and proteomic profile of I.orientalis under acetic acid stress. The knowledge of the transcription and expression changes of the I.orientalis is expected to understand the tolerance mechanisms in I.orientalis and to guide traditional fermentation processes by Saccharomyces cerevisiae improving its high resistance to acetic acid stress.

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress

BackgroundPretreatment of lignocellulosic biomass generates different types of inhibitors (e.g., furfural and acetic acid), which could remarkably inhibit subsequent ethanol fermentation. Here, biochar as an additive in the fermentation broth was first applied to enhance ethanol production by Z. mobilis wild-type strain ZM4 in the presence of typical inhibitors.ResultsThis study showed that the biochar-mediated tolerance to furfural and acetic acid for the strain Z. mobilis ZM4 was the highest reported level, resulting in much higher ethanol productivity under stress conditions than that in non-treated conditions. Further analysis showed that adsorptive detoxification was not the controlling factor for enhanced ethanol production under stress conditions, attributed to its low removal of furfural (< 20%) and incapability of acetic acid removal. When biochar was filtered from the biochar-treated inhibitor-containing broth, it still showed enhanced ethanol production. Furthermore, Z. mobilis immobilized on biochar was also observed. Thus, biochar extracts in the fermentation broth and cell immobilization on biochar might be the controlling factors for enhanced ethanol production under stress conditions.ConclusionsThese results indicate that biochar-mediated enhanced ethanol fermentation (BMEEF) might be a promising strategy for ethanol production from lignocellulosic biomass.

Deletion of Atg22 gene contributes to reduce programmed cell death induced by acetic acid stress in Saccharomyces cerevisiae

BackgroundProgrammed cell death (PCD) induced by acetic acid, the main by-product released during cellulosic hydrolysis, cast a cloud over lignocellulosic biofuel fermented by Saccharomyces cerevisiae and became a burning problem. Atg22p, an ignored integral membrane protein located in vacuole belongs to autophagy-related genes family; prior study recently reported that it is required for autophagic degradation and efflux of amino acids from vacuole to cytoplasm. It may alleviate the intracellular starvation of nutrition caused by Ac and increase cell tolerance. Therefore, we investigate the role of atg22 in cell death process induced by Ac in which attempt is made to discover new perspectives for better understanding of the mechanisms behind tolerance and more robust industrial strain construction.ResultsIn this study, we compared cell growth, physiological changes in the absence and presence of Atg22p under Ac exposure conditions. It is observed that disruption and overexpression of Atg22p delays and enhances acetic acid-induced PCD, respectively. The deletion of Atg22p in S. cerevisiae maintains cell wall integrity, and protects cytomembrane integrity, fluidity and permeability upon Ac stress by changing cytomembrane phospholipids, sterols and fatty acids. More interestingly, atg22 deletion increases intracellular amino acids to aid yeast cells for tackling amino acid starvation and intracellular acidification. Further, atg22 deletion upregulates series of stress response genes expression such as heat shock protein family, cell wall integrity and autophagy.ConclusionsThe findings show that Atg22p possessed the new function related to cell resistance to Ac. This may help us have a deeper understanding of PCD induced by Ac and provide a new strategy to improve Ac resistance in designing industrial yeast strains for bioethanol production during lignocellulosic biofuel fermentation.

Effect of Hexavalent Chromium [Cr(VI)] on Phytoremediation Potential and Biochemical Response of Hybrid Napier Grass with and without EDTA Application.

Hexavalent chromium [Cr(VI)] contamination has become an emergent concern in China. Previous field investigations have found that hybrid Napier grass is widely distributed in Cr(VI) contaminated areas. This study investigated the phytoremediation potential and biochemical response of hybrid Napier grass (Pennisetum americanus L. × Pennisetum purpureum Schumach) grown in soil contaminated with Cr(VI) (0, 20, 40, and 60 mg kg−1) with and without Ethylene diamine tetra acetic acid (EDTA) (4 mM) application. The results indicated that root length, shoot height, dry weight, leaf area, chlorophyll, and photosystem II (PSII) parameters viz.; apparent electron transport rate (ETR), effective quantum yield of PSII (ΦPSⅡ), maximal PSII photochemical efficiency (Fv/Fm), potential activity of PSII (Fv/Fo), photochemical quenching (qP), and non-photochemical quenching (qN) decreased with the increasing Cr(VI) concentration. EDTA application further aggravated reduction of dry biomass and photosystem II. The concentration and the accumulation of Cr in shoot and root, and both the bioaccumulation factor (BAF) and transfer factor (TF) increased with increasing Cr(VI) concentrations and further enhanced with EDTA application. Though the Cr(VI) and Ethylene diamine tetra acetic acid (EDTA) stress reduced tolerance, but, even at highest Cr(VI) concentration, plant could exhibited strong resistance, as evidenced by increase in superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Hybrid Napier grass, due to its BAF > 1 and a TF < 1, would be applicable for Cr phytostabilization. Moreover, limiting metal transport to aerial parts of plant would prevent animal’s ingestion, restrict soil mobility, and consequently reduce transmission across the food chain.

Optimization of low-temperature energy-efficient pretreatment for enhanced saccharification and fermentation of Conocarpus erectus leaves to produce ethanol using Saccharomyces cerevisiae

Leaves of Conocarpus erectus are potential feedstock for bioethanol production due to their lower lignin and higher cellulosic content and are among one of the least explored biomass sources. The present study reported a low-temperature (90 °C) energy-efficient pretreatment method for the release of fermentable sugars from C. erectus leaves and its conversion into bioethanol by using industrial and lab yeast strains. Optimization of process parameters was performed by monitoring glucose utilization and ethanol production. The maximum glucose concentration of 12 g L−1 and 19.75 g L−1 were obtained after the low-temperature (90 °C) pretreatment and enzymatic saccharification, respectively. The optimum enzymatic assay conditions were with 10% biomass and enzyme loading of 30 FPU/g cellulose, resulting in the release of ~ 20 g L−1 of glucose from pretreated biomass which is equivalent to the glucose concentration in standard growth media. Hydrolysate fermentation by S. cerevisiae strains SPSC01, ER, YB2625, and 6525 exhibited highest ethanol yields of 0.47 g g−1 and 0.46 g g−1 which is the highest ever reported for C. erectus even under acetic acid stress, where a fermentation efficiency of 92.3% was achieved. The present study may lead to establishing low-cost, energy-efficient biological conversion of C. erectus to ethanol.

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

AbstractLignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.

Improvement of inhibitor tolerance in Saccharomyces cerevisiae by overexpression of the quinone oxidoreductase family gene YCR102C.

Budding yeast Saccharomyces cerevisiae is widely used for lignocellulosic biorefinery. However, its fermentation efficiency is challenged by various inhibitors (e.g. weak acids, furfural) in the lignocellulosic hydrolysate, and acetic acid is commonly present as a major inhibitor. The effects of oxidoreductases on the inhibitor tolerance of S. cerevisiae have mainly focused on furfural and vanillin, whereas the influence of quinone oxidoreductase on acetic acid tolerance is still unknown. In this study, we show that overexpression of a quinone oxidoreductase-encoding gene, YCR102C, in S. cerevisiae, significantly enhanced ethanol production under acetic acid stress as well as in the inhibitor mixture, and also improved resistance to simultaneous stress of 40°C and 3.6 g/L acetic acid. Increased catalase activities, NADH/NAD+ ratio and contents of several metals, especially potassium, were observed by YCR102C overexpression under acetic acid stress. To our knowledge, this is the first report that the quinone oxidoreductase family protein is related to acid stress tolerance. Our study provides a novel strategy to increase lignocellulosic biorefinery efficiency using yeast cell factory.

Characterization and comparative analysis of toxin-antitoxin systems in Acetobacter pasteurianus.

Bacterial toxin-antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three putative type II TA candidates from Acetobacter pasteurianus and investigate the profile of type II TA systems in the genus Acetobacter. Based on the gene structure and activity detection, two-pairs loci were identified as the canonical hicAB and higAB TA systems, respectively, and DB34_01190-DB34_01195 as a putative new one without a canonical TA architecture. Physiologically, the expression of the three pairs conferred E. coli with additional plasmid maintenance and survival when under acetic acid stress. Chromosomal TA systems can be horizontally transferred within an ecological vinegar microbiota by co-option, and there was a tendency for toxin module loss. The antitoxin retention in the genome is suggested to have a broad role in bacterial physiology. Furthermore, A. pasteurianus strains, universally domesticated and used for industrial vinegar fermentation, showed a higher number of type II TA loci compared to the host-associated ones. The amount of TA loci per genome showed little positive relationship to insertion sequences, although its prevalence was species-associated, to the extent of even being strain-associated. The TA system is a candidate of studying the resistant mechanistic network, the TAs-dependent translatome affords a real-time profile to explore stress adaptation of A. pasteurianus, promoting industrial development.

Insights on acetate-ethanol fermentation by hydrogen-producing Ethanoligenens under acetic acid accumulation based on quantitative proteomics

Ethanoligenens, a novel ethanologenic hydrogen-producing genus, is a representative fermenter in its unique acetate-ethanol fermentation and physiology. Acetic acid accumulation is one of major factors that affect H2-ethanol co-production. However, sufficient information is unavailable on the tolerance mechanisms of hydrogen-producing bacterium in acetic acid stress. The fermentation process of Ethanoligenens harbinense YUAN-3 was significantly slowed down in the selection stress of exogenous acetic acid. The maximum gas production rate of strain YUAN-3 decreased from 192.15 mL·(L-culture)−1·h−1 to 75.2 mL·(L-culture)−1·h−1 with increasing exogenous acetic acid from 0 mM to 30 mM, the batch fermentation period was correspondingly expanded from 66 h to 136 h. Through iTRAQ-based quantitative proteomic approach, 78, 121 and 216 proteins were differentially expressed after strain YUAN-3 was cultured in the medium supplemented with exogenous acetic acid of 10 mM, 20 mM and 30 mM. The up-regulated proteins were mainly involved in β-alanine and pyrimidine metabolism, oxidative stress response, while the down-regulated proteins mainly participated in phosphotransferase system (PTS), fructose and mannose metabolism, phosphate uptake, ribosome, and flagellar assembly. These proteins help to maintain balance between fermentation process and alleviation of intracellular acidification in strain YUAN-3. The study indicated that response to acetic acid stress in strain YUAN-3 was a complex process, which involved multiple metabolic pathways. Reductive pyrimidine catabolic pathway played an important role in the acetic acid resistance of E. harbinense.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes

BackgroundYeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study.ResultsThree genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool “AXP” ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress.ConclusionWe proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

Molecular approaches to understand the effect of acetic acid in uropathogenic E. coli

Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent. To do this, we grew a high-density transposon library in uropathogenic E. coli EO499 serotype 131 in M9 media at pH 7 and pH 5.5 with acetic acid concentrations of 40 mM and 4 mM, respectively, or without added acetic acid. Sequencing libraries were generated from total bacterial populations after growth, and sequenced using a transposon-specific primer to generate positions and frequencies for each transposon. By comparing numbers of reads before and after the stress, we identified candidate genes where transposon inserts led to a decrease of fitness under acetic acid stress. Eight of these were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in time course competitions at pH 5.5 with acetic acid. This may due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library for the determination of relative fitness. The results will be presented.

Engineered Zymomonas mobilis tolerant to acetic acid and low pH via multiplex atmospheric and room temperature plasma mutagenesis

BackgroundCellulosic biofuels are sustainable compared to fossil fuels. However, inhibitors, such as acetic acid generated during lignocellulose pretreatment and hydrolysis, would significantly inhibit microbial fermentation efficiency. Microbial mutants able to tolerate high concentration of acetic acid are needed urgently to alleviate this inhibition.ResultsZymomonas mobilis mutants AQ8-1 and AC8-9 with enhanced tolerance against acetic acid were generated via a multiplex atmospheric and room temperature plasma (mARTP) mutagenesis. The growth and ethanol productivity of AQ8-1 and AC8-9 were both improved in the presence of 5.0–8.0 g/L acetic acid. Ethanol yield reached 84% of theoretical value in the presence of 8.0 g/L acetic acid (~ pH 4.0). Furthermore, a mutant tolerant to pH 3.5, named PH1-29, was generated via the third round of ARTP mutagenesis. PH1-29 showed enhanced growth and ethanol production under both sterilized/unsterilized conditions at pH 4.0 or 3.5. Intracellular NAD levels revealed that mARTP mutants could modulate NADH/NAD+ ratio to respond to acetic acid and low pH stresses. Moreover, genomic re-sequencing revealed that eleven single nucleic variations (SNVs) were likely related to acetic acid and low pH tolerance. Most SNVs were targeted in regions between genes ZMO0952 and ZMO0956, ZMO0152 and ZMO0153, and ZMO0373 and ZMO0374.ConclusionsThe multiplex mutagenesis strategy mARTP was efficient for enhancing the tolerance in Z. mobilis. The ARTP mutants generated in this study could serve as potential cellulosic ethanol producers.