Butanol Stress Research Articles

Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Native SodB Overexpression of Synechocystis sp. PCC 6803 Improves Cell Growth Under Alcohol Stresses Whereas Its Gpx2 Overexpression Impacts on Growth Recovery from Alcohol Stressors.

To overcome the limited resistance to alcohol stress, genetically engineered Synechocystis sp. PCC 6803 strains with overexpressions of genes related with the ROS detoxification system (sodB and gpx2, which encode superoxide dismutase and glutathione peroxidase, respectively) were developed. Three engineered strains including a sodB-overexpressing strain (OE + S), a gpx2-overexpressing strain (OE + G), and a sodB/gpx2-overexpressing strain (OE + SG) grew similarly as wild-type control under normal condition. When compared to wild-type control, OE + S and OE + SG strains grew faster for 4days under 2.0% (v/v) ethanol and 0.3% (v/v) n-butanol conditions, as well as having higher chlorophyll a levels. On the other hand, the prominent growth recovery of OE + G and OE + SG was noted within 4days in normal BG11 medium after treating cells with high alcohol stresses for 1h, in particular 15% ethanol and 2.5% n-butanol. Under 4days of recovery from butanol stress, specific levels of intracellular pigments including chlorophyll a and carotenoids were dramatically increased in all modified strains. The overexpression of antioxidant genes then revealed a significant improvement of alcohol tolerance in Synechocystis sp. PCC 6803.

Response characteristics of the membrane integrity and physiological activities of the mutant strain Y217 under exogenous butanol stress.

Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

Butanol Tolerance of Lactiplantibacillus plantarum: A Transcriptome Study.

Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

Effects of Carbon Ion Beam Irradiation on Butanol Tolerance and Production of Clostridium acetobutylicum.

Clostridium acetobutylicum (C. acetobutylicum) has considerable potential for use in bioenergy development. Owing to the repeated use of traditional mutagenesis methods, the strains have developed a certain tolerance. The rheology of the bioprocess and the downstream processing of the product heavily depend on the ability of C. acetobutylicum mutants to produce butanol. Carbon ion beam irradiation has advantages over traditional mutation methods for fermentative production because of its dose conformity and superb biological effectiveness. However, its effects on the specific productivity of the strains have not been clearly understood. In this study, we screened five mutants through carbon ion beam irradiation; mutant Y217 achieved a butanol-production level of 13.67 g/L, exceeding that of wild-type strain ATCC 824 (i.e., 9.77 g/L). In addition, we found that the mutant maintained normal cell membrane integrity under the stimulation of 15 g/L butanol, whereas the intracellular macromolecules of wild-type strain ATCC 824 leaked significantly. Subsequently, we used the response surface methodology (RSM) to determine if the mutant cell membrane integrity improved the butanol tolerance. We verified that with the addition of butanol, the mutant could be fermented to produce 8.35 g/L butanol, and the final butanol concentration in the fermentation broth could reach 16.15 g/L. In this study, we proved that under butanol stress, mutant Y217 features excellent butanol production and tolerance and cell membrane integrity and permeability; no prior studies have attempted to do so. This will serve as an interesting and important illustration of the complexity of genetic control of the irradiation mutation of C. acetobutylicum strains. It may also prove to be useful in the bioengineering of strains of the mutant for use in the predevelopment stage.

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance.

N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in Escherichia coli.

Producing high concentrations of biobutanol is challenging, primarily because of the toxicity of butanol toward cells. In our previous study, several butanol tolerance-promoting genes were identified from butanol-tolerant Escherichia coli mutants and inactivation of the transcriptional regulator factor Rob was shown to improve butanol tolerance. Here, the butanol tolerance characteristics and mechanism regulated by inactivated Rob are investigated. Comparative transcriptome analysis of strain DTrob, with a truncated rob in the genome, and the control BW25113 revealed 285 differentially expressed genes (DEGs) to be associated with butanol tolerance and categorized as having transport, localization, and oxidoreductase activities. Expression of 25 DEGs representing different functional categories was analyzed by quantitative reverse transcription PCR (qRT-PCR) to assess the reliability of the RNA-Seq data, and 92% of the genes showed the same expression trend. Based on functional complementation experiments of key DEGs, deletions of glgS and yibT increased the butanol tolerance of E. coli, whereas overexpression of fadB resulted in increased cell density and a slight increase in butanol tolerance. A metabolic network analysis of these DEGs revealed that six genes (fadA, fadB, fadD, fadL, poxB, and acs) associated with acetyl-CoA production were significantly upregulated in DTrob, suggesting that Rob inactivation might enhance butanol tolerance by increasing acetyl-CoA. Interestingly, DTrob produced more acetate in response to butanol stress than the wild-type strain, resulting in the upregulation expression of some genes involved in acetate metabolism. Altogether, the results of this study reveal the mechanism underlying increased butanol tolerance in E. coli regulated by Rob inactivation.

The biological mechanisms of butanol tolerance and the application of solvent‐tolerant bacteria for environmental protection

AbstractToxicity caused by the accumulation of butanol in fermentation media is an important factor limiting the concentration of butanol. There is currently no systematic research in place investigating the butanol tolerance mechanism of bacteria such as Clostridium acetobutylicum, which adapts to butanol stress and regulates its growth and metabolism. Here, research results related to the butanol tolerance of C. acetobutylicum are reviewed to understand the molecular basis of changes in butanol‐tolerant strains. Organic solvent‐tolerant bacteria play an important role in the fields of biofuel production, enzyme preparation and bioremediation. An analysis of limitations of the application of organic solvent‐tolerant bacteria has revealed that future research should focus on combining the microbial tolerance phenotype with specific utilization to achieve an optimal balance between organic solvent tolerance and production. This review serves as a reference for the improvement and engineering of strains that tolerate organic solvents. © 2019 Society of Chemical Industry

Accumulation of sugars and nucleosides in response to high salt and butanol stress in 1-butanol producing Synechococcus elongatus

1-Butanol production using photosynthetic organisms such as cyanobacteria has garnered interest among researchers due to its high potential as a sustainable biofuel. Previously, the cyanobacterium Synechococcus elongatus PCC 7942 was engineered to produce 1-butanol through the introduction of a modified CoA-dependent pathway. S.elongatus strain DC11, a high producer of 1-butanol, was constructed based on metabolomics-assisted strain engineering. DC11 can reach a production titer of 418.7mg/L in 6 days, cutting the production time in half compared to the previously constructed DC7. Regardless, the final 1-butanol titer of DC11 was still low compared to other microbial hosts. Sensitivity towards 1-butanol of the producing strain has been known as one of main hurdles for improving cyanobacterial production system. Thus, to improve cyanobacterial-based 1-butanol production in the future, we employed the metabolomics approach to study the intrinsic effect of improved 1-butanol productivity in DC11. This study focused on metabolite profiling of DC11 using LC/MS/MS. Results showed that there is an accumulation of disaccharide-P and sucrose/trehalose in DC11 compared to the DC7. These metabolites were previously reported to have a role in salt and alcohol stress response in cyanobacteria and therefore, DC11 was subjected to 0.2M of NaCl and 1000mg/L of 1-butanol for further investigation. DC11 with stress treatment showed a more prominent accumulation of sugars and nucleosides compared to control. The results obtained from this study may be beneficial for future strain improvement strategies in S.elongatus, particularly addressing the metabolic response of this strain upon 1-butanol stress.

Identification of functional butanol-tolerant genes from Escherichia coli mutants derived from error-prone PCR-based whole-genome shuffling

BackgroundButanol is an important biofuel and chemical. The development of butanol-tolerant strains and the identification of functional butanol-tolerant genes is essential for high-yield bio-butanol production due to the toxicity of butanol.ResultsEscherichia coli BW25113 was subjected for the first time to error-prone PCR-based whole-genome shuffling. The resulting mutants BW1847 and BW1857 were found to tolerate 2% (v/v) butanol and short-chain alcohols, including ethanol, isobutanol, and 1-pentanol. The mutants exhibited good stability under butanol stress, indicating that they are potential host strains for the construction of butanol pathways. BW1847 had better butanol tolerance than BW1857 under 0–0.75% (v/v) butanol stress, but showed a lower tolerance than BW1857 under 1.25–2% (v/v) butanol stress. Genome resequencing and PCR confirmation revealed that BW1847 and BW1857 had nine and seven single nucleotide polymorphisms, respectively, and a common 14-kb deletion. Functional complementation experiments of the SNPs and deleted genes demonstrated that the mutations of acrB and rob gene and the deletion of TqsA increased the tolerance of the two mutants to butanol. Genome-wide site-specific mutated strains DT385 (acrB C1198T) and DT900 (rob AT686–7) also showed significant tolerance to butanol and had higher butanol efflux ability than the control, further demonstrating that their mutations yield an inactive protein that enhances butanol resistance characteristics.ConclusionsStable E. coli mutants with enhanced short alcohols and high concentrations of butanol tolerance were obtained through a rapid and effective method. The key genes of butanol tolerance in the two mutants were identified by comparative functional genomic analysis.

Tolerance against butanol stress by disrupting succinylglutamate desuccinylase in Escherichia coli.

Background: The four-carbon alcohol, butanol, is emerging as a promising biofuel and efforts have been undertaken to improve several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of butanol production. Although genomic tools (transcriptomics, proteomics, and metabolomics) have been widely used to investigate the cellular response to butanol stress, the existing knowledge of the molecular mechanisms involved in butanol tolerance is limited, and strain improvement is difficult due to the complexity of the regulatory network. Results: In this study, a butanol-tolerant Escherichia coli was constructed by disrupting gene astE (encoding succinylglutamate desuccinylase) to obtain higher butanol tolerance (increased by 34.6%). To clarify the tolerance mechanism, a metabolome analysis was also performed. As a result, a total of 73 metabolites (11 elevated and 62 decreased) were significantly changed. Most of the downregulated metabolites were mainly involved in the l-arginine degradation pathway, sulfate metabolic pathway, and 2-methylcitrate metabolic pathway. To further analyze the differential gene expression, a transcriptome was created. In total, 311 genes (113 upregulated and 198 downregulated) showed over a twofold difference and were associated with carbohydrate metabolism, energy metabolism, and ABC transporters. The integration of metabolomics and transcriptomics found that acid-activated glutaminase ybaS and the amino acid antiporter gadC were significantly up-regulated, but the levels of l-arginine and glutamate were not significantly increased and decreased. Therefore, the changes of amino acids between strains BW25113 and BW25113-ΔastE were measured by amino acid analysis. The ability of a mutant strain against acid stress was also measured by the growth experiment under various pH conditions in the absence of butanol. Conclusions: Based on the above experiments, it could be concluded that mutant BW25113-ΔastE mainly regulated intracellular pH-homeostasis to adapt to butanol stress, indicating the non-negligible impact of pH on microbial butanol tolerance, broadening our understanding of microbial butanol tolerance and providing a novel strategy for the rational engineering of a more robust butanol-producing host.

The significance of proline and glutamate on butanol chaotropic stress in Bacillus subtilis 168

BackgroundButanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities.ResultsUsing comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline–glutamate–arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested.ConclusionThis work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.

Isolation and characterization ofbutanol-tolerant Staphylococcus aureus.

A new solvent-tolerant species, Staphylococcus aureus, was isolated and characterized during the screening of butanol-tolerant microorganisms. Three isolates of S. aureus were obtained as contaminants during improvement of butanol tolerance of E. coli K12. Their cell dry weights were 135% that of K12 in the absence of butanol stress. S. aureus had a growth advantage over K12 when cultured with various concentrations of butanol. It can tolerate up to 3% (v/v) butanol, while most solventogenic bacteria can tolerate only 2% (v/v) butanol. The addition of 10-20g glucose/l enhanced its butanol tolerance. The relative cell biomass of the S. aureus was 71-306% that of E. coli under 5.5-10% (v/v) ethanol stress, indicating ethanol resistance. This is the first study to observe butanol-tolerant S. aureus. As this organism can be genetically manipulated, it could have a wide array of applications.

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.

Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP260.

This study aims to examine the effect of amino acid supplementation on solvent production by Clostridium beijerinckii during the acetone-butanol fermentation and to determine whether amino acids are involved in the acid tolerance response (ATR), which results in increased solvents. Fermentation studies with Cl. beijerinckii NCP 260 in limited-nitrogen media supplemented with glutamate, glutamine, lysine, proline, histidine or asparagine revealed that only glutamate, glutamine or histidine increased butanol titres comparable to control media. Acid survival tests at pH 5 showed that glutamate and histidine were effective in protecting Cl. beijerinckii cells against acid shock, and may be involved in the ATR. Using quantitative PCR, the transcription of the glutamine synthetase, nitrogen regulator and glutamate synthase operon (glnA-nitR-gltAB) was monitored during acid shock conditions, and expression of both the nitR and gltA genes was shown to be increased twofold. Glutamate and histidine specifically enhance the ATR in Cl. beijerinckii NCP 260, and the genes encoding glutamate synthase and the NitR regulator are both upregulated, predicted to lead to increased endogenous glutamate pools during acidogenesis. This may enhance the ATR and allow more viable cells to enter solventogenesis, thereby increasing butanol titres. Glutamine, glutamate and histidine may also afford protection from butanol stress directly. Using substrates naturally rich in glutamine, glutamate and histidine in industrial fermentations is a promising means to increase acid survival and solvent yields in solventogenic Clostridium.

Recent progress in biobutanol tolerance in microbial systems with an emphasis onClostridium

Biobased production of butanol promises a more sustainable route for industrial production. However, butanol toxicity remains a barrier for achieving high product titers. Investigation into butanol stress has shed some light on its modes of toxicity. Unfortunately, there still remain significant shortfalls in our understanding of the complex interactions of butanol with cells. To address this knowledge gap, a diverse range of tools have been employed to gain a better understanding of the adverse effects of butanol on the cell. These findings have lead to the identification of possible molecular mechanisms associated with butanol tolerance, which can be harnessed for future strain development efforts. This review focuses on recent efforts to address the toxicity of butanol in microbial producers and offers some perspectives on the future direction of this research sector.

High-Level Butanol Production from Cassava Starch by a Newly Isolated Clostridium acetobutylicum.

A new Clostridium acetobutylicum strain, exhibiting the ability to resist butanol stress and produce butanol, was identified and named GX01. Strain GX01 can use a wide variety of carbohydrates, especially cassava starch, to produce butanol. After the optimization of culture conditions, C. acetobutylicum GX01 could produce 27.3 g/L solvent, including 17.1 g/L butanol, 7.9 g/L acetone, and 2.3 g/L ethanol, from 100 g/L cassava flour and 3 g/L soybean meal. Furthermore, when its acetone-butanol-ethanol (ABE) fermentation was performed in 10- and 30-L bioreactors, the production of total solvent and butanol reached 29.2 and 18.3 g/L, respectively, and 28.8 and 18.8 g/L, respectively. Thus, the high level and stability of butanol production make strain GX01 a promising candidate for ABE fermentation using the low-cost cassava starch.

Changes in membrane plasmalogens of Clostridium pasteurianum during butanol fermentation as determined by lipidomic analysis.

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.

Elucidating butanol tolerance mediated by a response regulator Sll0039 in Synechocystis sp. PCC 6803 using a metabolomic approach.

Butanol is highly toxic to cyanobacterial cells, which could restrict the future application of this renewable system in producing carbon neutral biofuel butanol. To seek knowledge regarding butanol tolerance in cyanobacteria, a library of response regulator (RR) gene mutants of Synechocystis sp. PCC 6803 was screened. The results showed that the deletion mutant of an orphan RR-encoding genes ll0039 was more sensitive to butanol than the wild type, while complementation of the ∆slr1037 mutant with the sll0039 gene recovered its tolerance to the same level as the wild type, suggesting that the sll0039 gene was involved in the regulation of tolerance against butanol. Further analysis employing an integrated liquid chromatography-mass spectrometry (LC-MS)-based and gas chromatography-mass spectrometry (GC-MS)-based metabolomics was conducted to determine the possible regulatory network of butanol tolerance mediated by Sll0039. LC-MS analysis allowed the identification of several metabolites, such as adenosine 5'-diphosphate (ADP)-glucose, dihydroxyacetone phosphate (DHAP), D-ribose 5-phosphate (R5P), D-glucose 6-phosphate (G6P), D-fructose 6-phosphate (F6P), α-ketoglutaric acid (AKG), uridine 5'-diphospho (UDP)-glucose, and nicotinamide adenine dinucleotide phosphate (NADP), which were differentially regulated between the wild type and the ∆sll0039 mutant grown under butanol stress, while GC-MS analysis identified 1, 2, and 2 metabolic modules associated with the sll0039 gene deletion at 24, 48, and 72h, respectively, suggesting that they were under control directly or indirectly by Sll0039 RR. In addition, a metabolomic comparison of the metabolic responses to butanol stress was conducted in the ∆sll0039 mutant and the ∆slr1037 mutant previously found to be involved in butanol tolerance (Chen et al. Biotechnol Biofuels 7:89 2014a), and the results showed that the regulatory networks mediated by Sll0039 and Slr1037 could be functionally independent in Synechocystis. The results provided a metabolomic description of the butanol tolerance network regulated by Sll0039.

Comparative Proteomic and Metabolomic Analysis of Staphylococcus warneri SG1 Cultured in the Presence and Absence of Butanol

The complete genome of the solvent tolerant Staphylococcus warneri SG1 was recently published. This Gram-positive bacterium is tolerant to a large spectrum of organic solvents including short-chain alcohols, alkanes, esters and cyclic aromatic compounds. In this study, we applied a two-dimensional liquid chromatography (2D-LC) mass spectrometry (MS) shotgun approach, in combination with quantitative 2-MEGA (dimethylation after guanidination) isotopic labeling, to compare the proteomes of SG1 grown under butanol-free and butanol-challenged conditions. In total, 1585 unique proteins (representing 65% of the predicted open reading frames) were identified, covering all major metabolic pathways. Of the 967 quantifiable proteins by 2-MEGA labeling, 260 were differentially expressed by at least 1.5-fold. These proteins are involved in energy metabolism, oxidative stress response, lipid and cell envelope biogenesis, or have chaperone functions. We also applied differential isotope labeling LC-MS to probe metabolite changes in key metabolic pathways upon butanol stress. This is the first comprehensive proteomic and metabolomic study of S. warneri SG1 and presents an important step toward understanding its physiology and mechanism of solvent tolerance.