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Luzhou-flavor baijiu (LFB) is brewed by the combined action of various microorganisms, and its flavor is affected by the microbial community and the genes they express, but which genes are the key ones during LFB brewing is less clear. Based on our previous studies the genes ME2 and adhE were identified as key genes, but which role they play was also unknown. In this study functional microorganisms were screened based on the key genes ME2 and adhE, and they were identified to be Rummeliibacillus suwonensis, Clostridium tyrobutyricum and Lactobacillus buchneri. Then simulated fermentation experiments were carried out with the functional microorganisms, and during the fermentation process expression of the key genes and the amounts of the main flavors were detected to analyze the role of the key genes. The results showed that the key gene ME2 was significantly positively correlated with the contents of the main acids, however the key gene adhE and the formation of the main esters in the LFB brewing process was a significant positive correlation. This study verified the two key genes ME2 and adhE complement each other in the LFB brewing process, playing an important role in promoting the formation of flavor substances, and are very beneficial to improve the quality of LFB.

Inflammatory bowel disease (IBD) implies the chronic inflammation of the gastrointestinal tract, combined with systemic vascular manifestations. In IBD, the incidence of cardiovascular disease appears to be related to an increase of oxidative stress and endothelial dysfunction. Grape pomace contains high levels of anti-oxidant polyphenols that are able to counteract chronic inflammatory symptoms. The aim of this study was to determine whether grape pomace polyphenolic extract (GPE) was able to mitigate the overwhelming inflammatory response in enterocyte-like cells and to improve vascular function. Intestinal epithelial Caco-2 cells, grown in monolayers or in co-culture with endothelial cells (Caco-2/HMEC-1), were treated with different concentrations of GPE (1, 5, 10 µg/mL gallic acid equivalents) for 2 h and then stimulated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α for 16 h. Through multiple assays, the expression of intestinal and endothelial inflammatory mediators, intracellular reactive oxygen species (ROS) levels and NF-κB activation, as well as endothelial-leukocyte adhesion, were evaluated. The results showed that GPE supplementation prevented, in a concentration-dependent manner, the intestinal expression and release of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMP)-9 and MMP-2. In Caco-2 cells, GPE also suppressed the gene expression of several pro-inflammatory markers, such as IL-1β, TNF-α, macrophage colony-stimulating factor (M-CSF), C-X-C motif ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (COX)-2. The GPE anti-inflammatory effect was mediated by the inhibition of NF-κB activity and reduced intracellular ROS levels. Furthermore, transepithelial GPE suppressed the endothelial expression of IL-6, MCP-1, VCAM-1, and ICAM-1 and the subsequent adhesion of leukocytes to the endothelial cells under pro-inflammatory conditions. In conclusion, our findings suggest grape pomace as a natural source of polyphenols with multiple health-promoting properties that could contribute to the mitigation of gut chronic inflammatory diseases and improve vascular endothelial function.

Background Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Results Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Conclusions Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

Background Fructophilic lactic acid bacteria (FLAB) found in D-fructose rich niches prefer D-fructose over D-glucose as a growth substrate. They need electron acceptors for growth on D-glucose. The organisms share carbohydrate metabolic properties. Fructobacillus spp., Apilactobacillus kunkeei, and Apilactobacillus apinorum are members of this unique group. Here we studied the fructophilic characteristics of recently described species Apilactobacillus micheneri , Apilactobacillus quenuiae, and Apilactobacillus timberlakei . Results The three species prefer D-fructose over D-glucose and only metabolize D-glucose in the presence of electron acceptors. The genomic characteristics of the three species, i.e. small genomes and thus a low number of coding DNA sequences, few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, are characteristic of FLAB. The three species thus are novel members of FLAB. Reduction of genes involved in carbohydrate transport and metabolism in accordance with reduction of genome size were the common characteristics of the family Lactobacillaceae, but FLAB markedly reduced the gene numbers more than other species in the family. Pan-genome analysis of genes involved in metabolism displayed a lack of specific carbohydrate metabolic pathways in FLAB, leading to a unique cluster separation. Conclusions The present study expanded FLAB group. Fructose-rich environments have induced similar evolution in phylogenetically distant FLAB species. These are examples of convergent evolution of LAB.

Background Clostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures. Results To separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (P clo1313_2638 ::ldh* ∆ adhE ) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE , in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L −1 and was able to reach higher maximum OD 600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD 600 and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L −1 . For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L −1 at 45 and 50 °C compared to 55 °C. Conclusion Positive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.

Poly(lactic acid) (PLA) filaments have been the most used in fused deposition modeling (FDM) 3D printing. The filaments, based on PLA, are continuing to be developed to overcome brittleness, low heat resistance, and obtain superior mechanical performance in 3D printing. From our previous study, the binary blend composites from PLA and poly(butylene adipate-co-terephthalate) (PBAT) with nano talc (PLA/PBAT/nano talc) at 70/30/10 showed an improvement in toughness and printability in FDM 3D printing. Nevertheless, interlayer adhesion, anisotropic characteristics, and heat resistance have been promoted for further application in FDM 3D printing. In this study, binary and ternary blend composites from PLA/PBAT and poly(butylene succinate) (PBS) with nano talc were prepared at a ratio of PLA 70 wt. % and blending with PBAT or PBS at 30 wt. % and nano talc at 10 wt. %. The materials were compounded via a twin-screw extruder and applied to the filament using a capillary rheometer. PLA/PBAT/PBS/nano talc blend composites were printed using FDM 3D printing. Thermal analysis, viscosity, interlayer adhesion, mechanical properties, and dimensional accuracy of binary and ternary blend composite 3D prints were investigated. The incorporation of PBS-enhanced crystallinity of the blend composite 3D prints resulted in an improvement to mechanical properties, heat resistance, and anisotropic characteristics. Flexibility of the blend composites was obtained by presentation of PBAT. It should be noted that the core–shell morphology of the ternary blend influenced the reduction of volume shrinkage, which obtained good surface roughness and dimensional accuracy in the ternary blend composite 3D printing.

Previously, we showed that mice treated with cyclophosphamide (CTX) 4 days before intravenous injection of breast cancer cells had more cancer cells in the lung at 3 h after cancer injection than control counterparts without CTX. At 4 days after its injection, CTX is already excreted from the mice, allowing this pre-treatment design to reveal how CTX may modify the lung environment to indirectly affect cancer cells. In this study, we tested the hypothesis that the increase in cancer cell abundance at 3 h by CTX is due to an increase in the adhesiveness of vascular wall for cancer cells. Our data from protein array analysis and inhibition approach combined with in vitro and in vivo assays support the following two-prong mechanism. (1) CTX increases vascular permeability, resulting in the exposure of the basement membrane (BM). (2) CTX increases the level of matrix metalloproteinase-2 (MMP-2) in mouse serum, which remodels the BM and is functionally important for CTX to increase cancer abundance at this early stage. The combined effect of these two processes is the increased accessibility of critical protein domains in the BM, resulting in higher vascular adhesiveness for cancer cells to adhere. The critical protein domains in the vascular microenvironment are RGD and YISGR domains, whose known binding partners on cancer cells are integrin dimers and laminin receptor, respectively.

Enterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors.

Background Clostridium acetobutylicum possesses two homologous adhE genes, adhE1 and adhE2, which have been proposed to be responsible for butanol production in solventogenic and alcohologenic cultures, respectively. To investigate their contributions in detail, in-frame deletion mutants of each gene were constructed and subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic, and alcohologenic chemostat cultures. Results Under solventogenesis, compared to the control strain, only ΔadhE1 mutant exhibited significant changes showing decreased butanol production and transcriptional expression changes in numerous genes. In particular, adhE2 was over expressed (126-fold); thus, AdhE2 can partially replace AdhE1 for butanol production (more than 30 % of the in vivo butanol flux) under solventogenesis. Under alcohologenesis, only ΔadhE2 mutant exhibited striking changes in gene expression and metabolic fluxes, and butanol production was completely lost. Therefore, it was demonstrated that AdhE2 is essential for butanol production and thus metabolic fluxes were redirected toward butyrate formation. Under acidogenesis, metabolic fluxes were not significantly changed in both mutants except the complete loss of butanol formation in ΔadhE2, but numerous changes in gene expression were observed. Furthermore, most of the significantly up- or down-regulated genes under this condition showed the same pattern of change in both mutants. Conclusions This quantitative system-scale analysis confirms the proposed roles of AdhE1 and AdhE2 in butanol formation that AdhE1 is the key enzyme under solventogenesis, whereas AdhE2 is the key enzyme for butanol formation under acidogenesis and alcohologenesis. Our study also highlights the metabolic flexibility of C. acetobutylicum to genetic alterations of its primary metabolism.

K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe2+ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L−1 of ethanol (productivity of 0.50 g L−1 h−1 and yields of 0.15 g g−1) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L−1 (productivity of 0.59 g L−1 h−1 and yields of 0.16 g g−1) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.