Flor yeast immobilization in microbial biocapsules for Sherry wine production: microvinification approach.

Sherry wine is a pale-yellowish dry wine produced in Southern-Spain which features are mainly due to biological aging when the metabolism of biofilm-forming yeasts (flor yeasts) consumes ethanol (and other non-fermentable carbon sources) from a previous alcoholic fermentation, and produces volatile compounds such as acetaldehyde. To start aging and maintain the wine stability, a high alcohol content is required, which is achieved by the previous fermentation or by adding ethanol (fortification). Here, an alternative method is proposed which aims to produce a more economic, distinctive Sherry wine without fortification. For this, a flor yeast has been pre-acclimatized to glycerol consumption against ethanol, and later confined in a fungal-based immobilization system known as "microbial biocapsules", to facilitate its inoculum. Once aged, the wines produced using biocapsules and free yeasts (the conventional method) exhibited chemical differences in terms of acidity and volatile concentrations. These differences were evaluated positively by a sensory panel. Pre-acclimatization of flor yeasts to glycerol consumption was not successful but when cells were immobilized in fungal pellets, ethanol consumption was lower. We believe that immobilization of flor yeasts in microbial biocapsules is an economic technique that can be used to produce high quality differentiated Sherry wines.

Sir2 and Glycerol Underlie the Pro-Longevity Effect of Quercetin during Yeast Chronological Aging.

Quercetin (QUER) is a natural polyphenolic compound endowed with beneficial properties for human health, with anti-aging effects. However, although this flavonoid is commercially available as a nutraceutical, target molecules/pathways underlying its pro-longevity potential have yet to be fully clarified. Here, we investigated QUER activity in yeast chronological aging, the established model for simulating the aging of postmitotic quiescent mammalian cells. We found that QUER supplementation at the onset of chronological aging, namely at the diauxic shift, significantly increases chronological lifespan (CLS). Consistent with the antioxidant properties of QUER, this extension takes place in concert with a decrease in oxidative stress. In addition, QUER triggers substantial changes in carbon metabolism. Specifically, it promotes an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis due to improved catabolism of C2 by-products of yeast fermentation and glycerol. The former is attributable to the Sir2-dependent activity of phosphoenolpyruvate carboxykinase and the latter to the L-glycerol 3-phosphate pathway. Such a combined increased supply of gluconeogenesis leads to an increase in the reserve carbohydrate trehalose, ensuring CLS extension. Moreover, QUER supplementation to chronologically aging cells in water alone amplifies their long-lived phenotype. This is associated with intracellular glycerol catabolism and trehalose increase, further indicating a QUER-specific influence on carbon metabolism that results in CLS extension.

Aquaporin-7-Mediated Glycerol Permeability Is Linked to Human Sperm Motility in Asthenozoospermia and during Sperm Capacitation.

Osmoregulation plays a vital role in sperm function, encompassing spermatogenesis, maturation, and fertilization. Aquaglyceroporins, a subclass of aquaporins (AQPs), facilitate the transport of water and glycerol across the sperm membrane, with glycerol serving as an important substrate for sperm bioenergetics. This study aimed to elucidate the significance of AQP-mediated glycerol permeability in sperm motility. The presence and localization of AQP3 and AQP7 in human sperm were assessed using immunofluorescence. Subsequently, the glycerol permeability of spermatozoa obtained from normozoospermic individuals (n = 30) was measured, using stopped-flow light scattering, after incubation with specific aquaporin inhibitors targeting AQP3 (DFP00173), AQP7 (Z433927330), or general aquaglyceroporin (phloretin). Sperm from asthenozoospermic men (n = 30) were utilized to evaluate the AQP7-mediated glycerol permeability, and to compare it with that of normozoospermic men. Furthermore, hypermotile capacitated sperm cells were examined, to determine the AQP7 expression and membrane glycerol permeability. AQP3 was predominantly observed in the tail region, while AQP7 was present in the head, midpiece, and tail of human sperm. Our findings indicate that AQP7 plays a key role in glycerol permeability, as the inhibition of AQP7 resulted in a 55% decrease in glycerol diffusion across the sperm membrane. Importantly, this glycerol permeability impairment was evident in spermatozoa from asthenozoospermic individuals, suggesting the dysregulation of AQP7-mediated glycerol transport, despite similar AQP7 levels. Conversely, the AQP7 expression increased in capacitated sperm, compared to non-capacitated sperm. Hence, AQP7-mediated permeability may serve as a valuable indicator of sperm motility, and be crucial in sperm function.

Electrogenic Staphylococcus epidermidis colonizes nasal cavities and alleviates IL-6 progression induced by the SARS2-CoV nucleocapsid protein.

AIM: Certain probiotic bacteria have been shown to possess an immunomodulatory effect and a protective effect on influenza infections. Using the Staphylococcus epidermidis K1 colonized mice model, we assessed the effect of nasal administration of glycerol or flavin mononucleotide (FMN) on the production of interleukin (IL)-6 mediated by the severe acute respiratory syndrome coronavirus 2 (SARS2-CoV) nucleocapsid protein (NPP). METHODS AND RESULTS: FMN, one of the key electron donors for the generation of electricity facilitated by S. epidermidis ATCC 12228, was detected in the glycerol fermentation medium. Compared to the S. epidermidis ATCC 12228, the S. epidermidis K1 isolate showed significant expression of the electron transfer genes, including pyruvate dehydrogenase (pdh), riboflavin kinase (rk), 1,4-dihydroxy-2-naphthoate octaprenyltransferase (menA), and type II NADH quinone oxidoreductase (ndh2). Institute of cancer research (ICR) mice were intranasally administered with S. epidermidis K1 with or without pretreatment with riboflavin kinase inhibitors, then nasally treated with glycerol or FMN before inoculating the NPP. Furthermore, J774A.1 macrophages were exposed to NPP serum and then treated with NPP of SARS2-CoV. The IL-6 levels in the bronchoalveolar lavage fluid (BALF) of mice and macrophages were quantified using a mouse IL-6 enzyme-linked immunosorbent assay kit. CONCLUSIONS: Here, we report that nasal administration of NPP strongly elevates IL-6 levels in both BALF and J774A.1 macrophages. It is worth noting that NPP-neutralizing antibodies can decrease IL-6 levels in macrophages. The nasal administration of glycerol or FMN to S. epidermidis K1-colonized mice results in a reduction of NPP-induced IL-6 production.

Identification of Salivary Metabolic Signatures Associated with Primary Sjögren's Disease.

Sjögren's disease (SjD) is the second most prevalent autoimmune disorder that involves chronic inflammation of exocrine glands. Correct diagnosis of primary SjD (pSjD) can span over many years since disease symptoms manifest only in advanced stages of salivary and lachrymal glandular destruction, and consensus diagnostic methods have critical sensitivity and selectivity limitations. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the composition of metabolites in unstimulated saliva samples from 30 pSjD subjects and 30 participants who do not have Sjögren's disease (non-Sjögren's control group, NS-C). Thirty-four metabolites were quantified in each sample, and analysis was conducted on both non-normalized (concentration) and normalized metabolomics data from all study participants (ages 23-78) and on an age-restricted subset of the data (ages 30-70) while applying false discovery rate correction in determining data significance. The normalized data of saliva samples from all study participants, and of the age-restricted subset, indicated significant increases in the levels of glucose, glycerol, taurine, and lactate, as well as significant decreases in the levels of 5-aminopentanoate, acetate, butyrate and propionate, in subjects with pSjD compared to subjects in the NS-C group. Additionally, a significant increase in choline was found only in the age-restricted subset, and a significant decrease in fucose was found only in the whole study population in normalized data of saliva samples from the pSjD group compared to the NS-C group. Metabolite concentration data of saliva samples from all study participants, but not from the age-restricted subset, indicated significant increases in the levels of glucose, glycerol, taurine, and lactate in subjects with pSjD compared to controls. The study showed that NMR metabolomics can be implemented in defining salivary metabolic signatures that are associated with disease status, and can contribute to differential analysis between subjects with pSjD and those who are not affected with this disease, in the clinic.

Loss of function of Hog1 improves glycerol assimilation in Saccharomyces cerevisiae.

We previously isolated a mutant of Saccharomyces cerevisiae strain 85_9 whose glycerol assimilation was improved through adaptive laboratory evolution. To investigate the mechanism for this improved glycerol assimilation, genome resequencing of the 85_9 strain was performed, and the mutations in the open reading frame of HOG1, SIR3, SSB2, and KGD2 genes were found. Among these, a frameshift mutation in the HOG1 open reading frame was responsible for the improved glycerol assimilation ability of the 85_9 strain. Moreover, the HOG1 gene disruption improved glycerol assimilation. As HOG1 encodes a mitogen-activated protein kinase (MAPK), which is responsible for the signal transduction cascade in response to osmotic stress, namely the high osmolarity glycerol (HOG) pathway, we investigated the effect of the disruption of PBS2 gene encoding MAPK kinase for Hog1 MAPK on glycerol assimilation, revealing that PBS2 disruption can increase glycerol assimilation. These results indicate that loss of function of Hog1 improves glycerol assimilation in S. cerevisiae. However, single disruption of the SSK2, SSK22 and STE11 genes encoding protein kinases responsible for Pbs2 phosphorylation in the HOG pathway did not increase glycerol assimilation, while their triple disruption partially improved glycerol assimilation in S. cerevisiae. In addition, the HOG1 frameshift mutation did not improve glycerol assimilation in the STL1-overexpressing RIM15 disruptant strain, which was previously constructed with high glycerol assimilation ability. Furthermore, the effectiveness of the HOG1 disruptant as a bioproduction host was validated, indicating that the HOG1 CYB2 double disruptant can produce L-lactic acid from glycerol.

An artificial multienzyme cascade for the whole-cell synthesis of rare ketoses from glycerol.

PURPOSE: In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. METHODS: To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. RESULTS: The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l-1 only from the sole carbon source glycerol. CONCLUSION: This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.

Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator.

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.

A genome-scale metabolic model of the effect of dissolved oxygen on 1,3-propanediol fermentation by Klebsiella pneumoniae.

Although 1,3-propanediol (1,3-PD) is usually considered an anaerobic fermentation product from glycerol by Klebsiella pneumoniae, microaerobic conditions proved to be more conducive to 1,3-PD production. In this study, a genome-scale metabolic model (GSMM) specific to K. pneumoniae KG2, a high 1.3-PD producer, was constructed. The iZY1242 model contains 2090 reactions, 1242 genes and 1433 metabolites. The model was not only able to accurately characterise cell growth, but also accurately simulate the fed-batch 1,3-PD fermentation process. Flux balance analyses by iZY1242 was performed to dissect the mechanism of stimulated 1,3-PD production under microaerobic conditions, and the maximum yield of 1,3-PD on glycerol was 0.83 mol/mol under optimal microaerobic conditions. Combined with experimental data, the iZY1242 model is a useful tool for establishing the best conditions for microaeration fermentation to produce 1,3-PD from glycerol in K. pneumoniae.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Effect of crude glycerin supplementation via drinking water on feed intake, water intake and productive performance of dairy ewes.

This study was performed to determine the effects of crude glycerin (CG) supplementation in drinking water on DM and nutrient intake, milk production, milk composition, and serum glucose. Twenty multiparous Lacaune × East Friesian ewes were randomly distributed into four dietary treatments throughout the lactation cycle. Treatments consisted of doses of CG supplementation via drinking water as follows: (1) no CG supplementation, (2) 15.0 g CG/kg DM, (3) 30.0 g CG/kg DM, and (4) 45.0 g CG/kg DM. DM and nutrient intake were reduced linearly with CG supplementation. CG linearly reduced water intake when expressed as kg d-1. However, no effect of CG was observed when it was expressed as a percentage of body weight or metabolic body weight. The water to DM intake ratio was increased linearly with CG supplementation. No effect of CG doses on serum glucose was observed. The production of standardized milk decreased linearly with the experimental doses of CG. Protein, fat, and lactose yield were linearly reduced with the experimental doses of CG. Milk urea concentration was quadratically increased with CG doses. Feed conversion was quadratically increased by treatments during the pre-weaning period (P < 0.05), in which the worst values were observed when the ewes were supplemented with 15 and 30 g CG/kg DM. The N-efficiency was linearly increased with CG supplementation in drinking water. Our results suggest that dairy sheep can be supplemented with CG up to 15 g/kg DM in drinking water. Greater doses are not beneficial for feed intake, milk production, and the yield of milk components.

Integrated multiomics analysis reveals changes in liver physiological function in Aqp9 gene knockout mice.

Aquaporin 9 (AQP9) is the main channel by which blood glycerol enters the liver, where it plays key roles in osmotic pressure regulation and energy metabolism. Previous studies have shown that AQP9 is involved in the pathogenesis of many liver diseases. In this study, we aimed to clarify the role of AQP9 in maintaining the physiological environment of the liver using Aqp9-/- mice. We constructed Aqp9 knockout mice and used comprehensive multiomics analysis to elucidate the potential molecular effects of AQP9 expression on liver tissue. Knockout of Aqp9 reduced mouse body weight by affecting glycerol metabolism and led to hepatocyte death and inflammatory cell infiltration, which was confirmed by transcriptomics, proteomics and metabolomics. Moreover, knockout of Aqp9 triggered immune and inflammatory responses, leading to scattered and mild liver cell pyroptosis and compensatory liver cell proliferation.

Set up and validation of a sensitive method to quantify prostaglandins, prostaglandin-glycerol esters and prostaglandin-ethanolamides, as well as their respective precursors.

Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively. The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

Time-resolved transcriptomic profile of oleaginous yeast Rhodotorula mucilaginosa during lipid and carotenoids accumulation on glycerol.

The study reports the exploration of the transcriptome landscape of the red oleaginous yeast Rhodotorula mucilaginosa IIPL32 coinciding with the fermentation kinetics of the yeast cultivated in a two-stage fermentation process to exploit the time-series approach to get the complete transcripts picture and reveal the persuasive genes for fatty acid and terpenoid synthesis. The finding displayed the molecular drivers with more than 2-fold upregulation in the nitrogen-limited stage than in the nitrogen-excess stage. The rate-limiting diphosphomevalonate decarboxylase, acetylCoA-citrate lyase, and acetyl-CoA C-acetyltransferase were significant in controlling the metabolic flux in the synthesis of reduced compounds, and acetoacetyl-CoA synthase, 3-ketoacyl-acyl carrier-protein reductase, and ß-subunit enoyl reductase catalyze the key starting steps of lipids or terpenoid synthesis. The last two catalyze essential reduction steps in fatty acid synthesis. These enzymes would be the prime targets for the metabolic engineering of the oleaginous yeast for enhanced fatty acids and terpenoid production.

High production of poly(3-hydroxybutyrate) in Escherichia coli using crude glycerol.

Poly(3-hydroxybutyrate) (PHB) is a prominent bio-plastic and recognized as the potential replacement of petroleum-derived plastics. To make PHB cost-effective, the production scheme based on crude glycerol was developed using Escherichia coli. The heterogeneous synthesis pathway of PHB was introduced into the E. coli strain capable of efficiently utilizing glycerol. The central metabolism that links to the synthesis of acetyl-CoA and NADPH was further reprogrammed to improve the PHB production. Key genes were targeted for manipulation, involving those in glycolysis, the pentose phosphate pathway, and the tricarboxylic cycle. As a result, the engineered strain gained a 22-fold increase in the PHB titer. Finally, the fed-batch fermentation was conducted with the producer strain to give the PHB titer, content, and productivity reaching 36.3 ± 3.0 g/L, 66.5 ± 2.8%, and 1.2 ± 0.1 g/L/h, respectively. The PHB yield on crude glycerol accounts for 0.3 g/g. The result indicates that the technology platform as developed is promising for the production of bio-plastics.

Electrobiochemical skills of Pseudomonas aeruginosa species that produce pyocyanin or pyoverdine for glycerol oxidation in a microbial fuel cell.

Pseudomonas aeruginosa can produce pigments, which mediate external electron transfer (EET). Depending on the mediator, this species can be explored in bioelectrosystems to harvest energy or to obtain chemicals from residual organic compounds. This study has compared the performance of microbial fuel cells (MFCs) inoculated with a Pseudomonas aeruginosa isolate, namely EW603 or EW819, which produce pyocyanin and pyoverdine, respectively. The efficiency of these MFCs in glycerol, a typical residue of biodiesel production, were also compared. The MFCs exhibited different performances. The maximum voltage was 411 and 281 mV m2, the power density was 40.1 and 21.3 mW m-2, and the coulombic efficiency was 5.16 and 1.49% for MFC-EW603 and MFC-EW819, respectively. MFC-EW603 and MFC-EW819 achieved maximum current at 560 and 2200 Ω, at 141.2 and 91.3 mA m-2, respectively. When the system was operated at the respective maximum current output, MFC-EW603 consumed the total glycerol content (11 mmol L-1), and no products could be detected after 50 h. In turn, acetic and butyric acids were detected at the end of MFC-EW819 operation (75 h). The results suggested that P. aeruginosa metabolism can be steered in the MFC to generate current or microbial products depending on the pigment-producing strain and the conditions applied to the system, such as the external resistance. In addition, gene cluster pathways related to phenazine production (phzA and phzB) and other electrogenic-related genes (mexGHI-opmB) were identified in the strain genomes, supporting the findings. These results open new possibilities for using glycerol in bioelectrochemical systems.

Fate of carbon in synthetic media fermentations containing Metschnikowia pulcherrima or Meyerozyma guilliermondii in the presence and absence of Saccharomyces cerevisiae.

While sequentially inoculating non-Saccharomyces yeasts with Saccharomyces cerevisiae can lower the alcohol contents of wine, the abilities of these yeasts to utilize/produce ethanol or generate other byproducts remained unclear. Metschnikowia pulcherrima or Meyerozyma guilliermondii were inoculated into media with or without S. cerevisiae to assess byproduct formation. Both species metabolized ethanol in a yeast-nitrogen-base medium but produced the alcohol in a synthetic grape juice medium. In fact, Mt. pulcherrima and My. guilliermondii generated less ethanol per gram of metabolized sugar (0.372 and 0.301 g/g, respectively) compared to S. cerevisiae (0.422 g/g). Sequentially inoculating each non-Saccharomyces species with S. cerevisiae into grape juice media achieved up to 3.0% v/v alcohol reduction compared to S. cerevisiae alone while producing variable glycerol, succinic acid, and acetic acid concentrations. However, neither non-Saccharomyces yeasts released appreciable CO2 under fermentative conditions regardless of incubation temperature. Despite equivalent peak populations, S. cerevisiae produced more biomass (2.98 g/L) than the non-Saccharomyces yeasts while sequential inoculations yielded higher biomass with Mt. pulcherrima (3.97 g/L) but not My. guilliermondii (3.03 g/L). To reduce ethanol concentrations, these non-Saccharomyces species may metabolize ethanol and/or produce less from metabolized sugars compared to S. cerevisiae but also divert carbon towards glycerol, succinic acid, and/or biomass.

In-depth analysis of erythrose reductase homologs in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.

Activation of CWI pathway through high hydrostatic pressure, enhancing glycerol efflux via the aquaglyceroporin Fps1 in Saccharomyces cerevisiae.

The fungal cell wall is the initial barrier for the fungi against diverse external stresses, such as osmolarity changes, harmful drugs, and mechanical injuries. This study explores the roles of osmoregulation and the cell-wall integrity (CWI) pathway in response to high hydrostatic pressure in the yeast Saccharomyces cerevisiae. We demonstrate the roles of the transmembrane mechanosensor Wsc1 and aquaglyceroporin Fps1 in a general mechanism to maintain cell growth under high-pressure regimes. The promotion of water influx into cells at 25 MPa, as evident by an increase in cell volume and a loss of the plasma membrane eisosome structure, activates the CWI pathway through the function of Wsc1. Phosphorylation of Slt2, the downstream mitogen-activated protein kinase, was increased at 25 MPa. Glycerol efflux increases via Fps1 phosphorylation, which is initiated by downstream components of the CWI pathway, and contributes to the reduction in intracellular osmolarity under high pressure. The elucidation of the mechanisms underlying adaptation to high pressure through the well-established CWI pathway could potentially translate to mammalian cells and provide novel insights into cellular mechanosensation.

Structural and functional insights into the flexible ß-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible ß-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the ß-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the ß-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible ß-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.