RESUMO
BACKGROUND: Microbial pdu and cob-cbi-hem gene clusters encode the key enzyme glycerol/diol dehydratase (PduCDE), which mediates the transformation of dietary nutrients glycerol and 1,2-propanediol (1,2-PD) to a variety of metabolites, and enzymes for cobalamin synthesis, a co-factor and shared good of microbial communities. It was the aim of this study to relate pdu as a multipurpose functional trait to environmental conditions and microbial community composition. We collected fecal samples from wild animal species living in captivity with different gut physiology and diet (n = 55, in total 104 samples), determined occurrence and diversity of pdu and cob-cbi-hem using a novel approach combining metagenomics with quantification of metabolic and genetic biomarkers, and conducted in vitro fermentations to test for trait-based activity. RESULTS: Fecal levels of the glycerol transformation product 1,3-propanediol (1,3-PD) were higher in hindgut than foregut fermenters. Gene-based analyses indicated that pduC harboring taxa are common feature of captive wild animal fecal microbiota that occur more frequently and at higher abundance in hindgut fermenters. Phylogenetic analysis of genomes reconstructed from metagenomic sequences identified captive wild animal fecal microbiota as taxonomically rich with a total of 4150 species and > 1800 novel species but pointed at only 56 species that at least partially harbored pdu and cbi-cob-hem. While taxonomic diversity was highest in fecal samples of foregut-fermenting herbivores, higher pduC abundance and higher diversity of pdu/cbi-cob-hem related to higher potential for glycerol and 1,2-PD utilization of the less diverse microbiota of hindgut-fermenting carnivores in vitro. CONCLUSION: Our approach combining metabolite and gene biomarker analysis with metagenomics and phenotypic characterization identified Pdu as a common function of fecal microbiota of captive wild animals shared by few taxa and stratified the potential of fecal microbiota for glycerol/1,2-PD utilization and cobalamin synthesis depending on diet and physiology of the host. This trait-based study suggests that the ability to utilize glycerol/1,2-PD is a key function of hindgut-fermenting carnivores, which does not relate to overall community diversity but links to the potential for cobalamin formation. Video Abstract.
Assuntos
Fezes , Fermentação , Microbioma Gastrointestinal , Glicerol , Metagenômica , Animais , Fezes/microbiologia , Glicerol/metabolismo , Metagenômica/métodos , Hidroliases/genética , Hidroliases/metabolismo , Propilenoglicóis/metabolismo , Vitamina B 12/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/enzimologia , Filogenia , Animais Selvagens/microbiologiaRESUMO
High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71 mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200 rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach.
Assuntos
Reatores Biológicos , Meios de Cultura , DNA Polimerase Dirigida por DNA , Escherichia coli , Pyrococcus furiosus , Proteínas Recombinantes , Pyrococcus furiosus/genética , Pyrococcus furiosus/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Reatores Biológicos/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Meios de Cultura/química , Glucose/metabolismo , Regiões Promotoras Genéticas , Glicerol/metabolismo , Lactose/metabolismoRESUMO
Yeast immobilization in beer fermentation has recently regained attention, due to the expansion of the craft beer market and the diversification of styles and flavors. The aim of this study was to evaluate the physiological differences between immobilized and free yeast cells with a focus on flavor-active compounds formation. Three strains of Saccharomyces spp. (SY025, SY067, SY001) were evaluated in both free and immobilized (using a cellulose-based support, referred as ImoYeast) forms during static batch fermentations of 12 °P malt extract. Immobilized cells showed higher glycerol (SY025, 40%; SY067, 53%; SY001, 19%) and biomass (SY025, 67%; SY067, 78%; SY001, 56%) yields than free cells. Conversely, free cells presented higher ethanol yield (SY025, 9%; SY067, 9%; SY001, 13%). Flavor-active compounds production exhibited significant alterations between immobilized and free cells systems, for all strains tested. Finally, a central composite design with varying initial biomass (X0) and substrate (S0) concentrations was conducted using strain SY025, which can be helpful to modulate the formation of one or more flavor-active compounds. In conclusion, yeast immobilization in the evaluated support resulted in flavor alterations that can be exploited to produce different beer styles.
Assuntos
Cerveja , Células Imobilizadas , Fermentação , Aromatizantes , Saccharomyces , Cerveja/microbiologia , Cerveja/análise , Saccharomyces/metabolismo , Aromatizantes/metabolismo , Células Imobilizadas/metabolismo , Biomassa , Etanol/metabolismo , Glicerol/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher D-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred D-fructose, D-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower D-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production.
Assuntos
Carbono , Celulose , Celulose/biossíntese , Celulose/metabolismo , Carbono/metabolismo , Acetobacteraceae/metabolismo , Acetobacteraceae/genética , Gluconatos/metabolismo , Glicerol/metabolismo , Manitol/metabolismoRESUMO
D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02â¯g/L with a maximum yield of 0.287â¯g/g.
Assuntos
Escherichia coli , Fermentação , Glicerol , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Glicerol/metabolismo , Reatores Biológicos/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , FrutoseRESUMO
Aquaporins (AQPs) are a family of integral membrane proteins that selectively transport water and glycerol across the cell membrane. Because AQPs are involved in a wide range of physiological functions and pathophysiological conditions, AQP-based therapeutics may have the broad potential for clinical utility, including for disorders of water and energy balance. However, AQP modulators have not yet been developed as suitable candidates for clinical applications. In this study, to identify potential modulators of AQPs, we screened 31 natural products by measuring the water and glycerol permeability of mouse erythrocyte membranes using a stopped-flow light scattering method. None of the tested natural compounds substantially affected the osmotic water permeability. However, several compounds considerably affected the glycerol permeability. Stichoposide C increased the glycerol permeability of mouse erythrocyte membranes, whereas rhizochalin decreased it at nanomolar concentrations. Immunohistochemistry revealed that AQP7 was the main aquaglyceroporin in mouse erythrocyte membranes. We further verified the effects of stichoposide C and rhizochalin on aquaglyceroporins using human AQP3-expressing keratinocyte cells. Stichoposide C, but not stichoposide D, increased AQP3-mediated transepithelial glycerol transport, whereas the peracetyl aglycon of rhizochalin was the most potent inhibitor of glycerol transport among the tested rhizochalin derivatives. Collectively, stichoposide C and the peracetyl aglycon of rhizochalin might function as modulators of AQP3 and AQP7, and suggests the possibility of these natural products as potential drug candidates for aquaglyceroporin modulators.
Assuntos
Aquagliceroporinas , Glicerol , Animais , Camundongos , Aquagliceroporinas/metabolismo , Humanos , Glicerol/metabolismo , Água/química , Água/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Aquaporina 3/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Aquaporinas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacosRESUMO
In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using Pseudomonas putida and Pseudomonas aeruginosa. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using Pseudomonas putida compared to Pseudomonas aeruginosa, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain.
Assuntos
Antibacterianos , Glicerol , Nanopartículas Metálicas , Poli-Hidroxialcanoatos , Pseudomonas aeruginosa , Pseudomonas putida , Prata , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas putida/metabolismo , Prata/química , Prata/farmacologia , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/química , Nanopartículas Metálicas/química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/biossíntese , Glicerol/química , Glicerol/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
In adipose tissue, reduced expression of the glycerol channel aquaporin 7 (AQP7) has been associated with increased accumulation of triglyceride. The present study determines the relative protein abundances of lipolytic enzymes, AQP7, and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in paired mesenteric and omental visceral adipose tissue (VAT) and abdominal and femoral subcutaneous adipose tissue (SAT) in women with either normal weight or upper-body obesity. No differences in the expression of hormone-sensitive lipase (HSL) or AQP7 were found between the two groups in the four depots. The expression of adipocyte triglyceride lipase (ATGL) and HSL were higher in omental VAT and femoral SAT than in mesenteric VAT in both groups of women. Similarly, AQP7 expression was higher in omental VAT than in mesenteric VAT. The expression of PEPCK-C was lower in omental VAT than in femoral SAT. No correlation between the expression of AQP7 and the mean adipocyte size was observed; however, the expression of PEPCK-C positively correlated with the mean adipocyte size. In conclusion, a depot-specific protein expression pattern was found for ATGL, HSL, AQP7, and PEPCK-C. The expression pattern supports that the regulation of AQP7 protein expression is at least in part linked to the lipolytic rate. Furthermore, the results support that the synthesis of glycerol-3-phosphate via glyceroneogenesis contributes to regulating triglyceride accumulation in white adipose tissue in women.
Assuntos
Aquaporinas , Glicerol , Gordura Intra-Abdominal , Obesidade , Gordura Subcutânea , Humanos , Feminino , Gordura Subcutânea/metabolismo , Aquaporinas/metabolismo , Aquaporinas/genética , Glicerol/metabolismo , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Adulto , Pessoa de Meia-Idade , Lipólise , Esterol Esterase/metabolismo , Esterol Esterase/genética , Lipase/metabolismo , Lipase/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Adipócitos/metabolismo , Triglicerídeos/metabolismo , AciltransferasesRESUMO
As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Glicerol , Peróxido de Hidrogênio , Boca , Streptococcus mutans , Glicerol/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Streptococcus mutans/crescimento & desenvolvimento , Boca/microbiologia , Streptococcus sanguis/metabolismo , Streptococcus sanguis/genética , Humanos , Biofilmes/crescimento & desenvolvimentoRESUMO
Developing utilization technologies for biomass resources, exploring their applications in the fields of energy and chemical engineering, holds significant importance for promoting sustainable development and constructing a green, low-carbon society. In this study, we designed a non-natural in vitro multi-enzyme system for converting glycerol and CO2 into L-aspartic acid (L-Asp). The coupled system utilized eight enzymes, including alditol oxidase (ALDO), catalase-peroxidase (CAT), lactaldehyde dehydrogenase (ALDH), glycerate 2-kinase (GK), phosphopyruvate hydratase (PPH), phosphoenolpyruvate carboxylase (PPC), L-aspartate dehydrogenase (ASPD), and polyphosphate kinase (PPK), to convert the raw materials into L-Asp in one-pot coupled with NADH and ATP regeneration. Under optimal reaction conditions, 18.6 mM of L-Asp could be produced within 2.0 h at a total enzyme addition of 4.85 mg/mL, demonstrating the high efficiency and productivity characteristics of the designed system. Our technological application provides new insights and methods for the development of biomass resource utilization technologies.
Assuntos
Ácido Aspártico , Dióxido de Carbono , Glicerol , Ácido Aspártico/metabolismo , Glicerol/metabolismo , Glicerol/química , Dióxido de Carbono/metabolismo , BiomassaRESUMO
Anaerobic microbial fermentations provide high product yields and are a cornerstone of industrial bio-based processes. However, the need for redox balancing limits the array of fermentable substrate-product combinations. To overcome this limitation, here we design an aerobic fermentative metabolism that allows the introduction of selected respiratory modules. These can use oxygen to re-balance otherwise unbalanced fermentations, hence achieving controlled respiro-fermentative growth. Following this design, we engineer and characterize an obligate fermentative Escherichia coli strain that aerobically ferments glucose to stoichiometric amounts of lactate. We then re-integrate the quinone-dependent glycerol 3-phosphate dehydrogenase and demonstrate glycerol fermentation to lactate while selectively transferring the surplus of electrons to the respiratory chain. To showcase the potential of this fermentation mode, we direct fermentative flux from glycerol towards isobutanol production. In summary, our design permits using oxygen to selectively re-balance fermentations. This concept is an advance freeing highly efficient microbial fermentation from the limitations imposed by traditional redox balancing.
Assuntos
Escherichia coli , Fermentação , Glucose , Glicerol , Ácido Láctico , Engenharia Metabólica , Escherichia coli/metabolismo , Glicerol/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Ácido Láctico/metabolismo , Oxirredução , Oxigênio/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Butanóis/metabolismo , AerobioseRESUMO
The ability of bacteria to colonize diverse environmental niches is often linked to their competence in biofilm formation. It depends on the individual characteristics of a strain, the nature of the colonized surface (abiotic or biotic), or the availability of certain nutrients. Pseudomonas donghuensis P482 efficiently colonizes the rhizosphere of various plant hosts, but a connection between plant tissue colonization and the biofilm formation ability of this strain has not yet been established. We demonstrate here that the potential of P482 to form biofilms on abiotic surfaces and the structural characteristics of the biofilm are influenced by the carbon source available to the bacterium, with glycerol promoting the process. Also, the type of substratum, polystyrene or glass, impacts the ability of P482 to attach to the surface. Moreover, P482 mutants in genes associated with motility or chemotaxis, the synthesis of polysaccharides, and encoding proteases or regulatory factors, which affect biofilm formation on glass, were fully capable of colonizing the root tissue of both tomato and maize hosts. Investigating the role of cellular factors in biofilm formation using these plant-associated bacteria shows that the ability of bacteria to form biofilm on abiotic surfaces does not necessarily mirror its ability to colonize plant tissues. Our research provides a broader perspective on the adaptation of these bacteria to various environments.
Assuntos
Biofilmes , Carbono , Pseudomonas , Biofilmes/crescimento & desenvolvimento , Pseudomonas/fisiologia , Pseudomonas/metabolismo , Pseudomonas/genética , Carbono/metabolismo , Raízes de Plantas/microbiologia , Rizosfera , Solanum lycopersicum/microbiologia , Zea mays/microbiologia , Vidro , Aderência Bacteriana , Glicerol/metabolismo , PoliestirenosRESUMO
1, 3-propanediol (1, 3-PDO) is an important diol with wide applications in the pharmaceutical, food, and cosmetics industries. In addition, 1, 3-PDO serves as a crucial monomer in the synthesis of polytrimethylene terephthalate, an important synthetic fiber material. Microbial conversion of renewable resources such as glucose into 1, 3-PDO has been industrialized due to its environmentally friendly, energy-efficient, safe, and sustainable characteristics. It serves as a successful case in the design and application of microbial cell factories for biochemicals. However, concerns such as food scarcity and climate change are driving the exploration of non-food, low-cost, and sustainable alternatives as biomanufacturing feedstocks. The biosynthesis of 1, 3-PDO from the C3 feedstock glycerol by microorganisms has been well studied. In recent years, increasing attention has been paid to the synthesis of 1, 3-PDO from C1 feedstocks such as methanol, which has higher energy density than glucose and glycerol. Several new artificial biosynthetic pathways have been proposed and validated, laying a foundation for the sustainable bioproduction of 1, 3-PDO. This article reviews the feedstock transition from C6 to C3 and C1 carbon sources for the microbial synthesis of 1, 3-PDO and discusses the strategies for reprogramming metabolic pathway to enhance 1, 3-PDO biosynthesis from different feedstocks. Finally, the development prospects of 1, 3-PDO bioproduction from C1 feedstocks are forecasted.
Assuntos
Carbono , Propilenoglicóis , Carbono/metabolismo , Propilenoglicóis/metabolismo , Glicerol/metabolismo , Microbiologia Industrial , Glucose/metabolismo , Engenharia Metabólica , Metanol/metabolismo , Vias Biossintéticas , Fermentação , Bactérias/metabolismoRESUMO
1, 3-propanediol is an important monomer for the production of polytrimethylene terephthalate (PTT). Currently, it is mainly produced by microbial fermentation, which, however, has low production efficiency. To address this problem, this study employed atmospheric room temperature plasma (ARTP) mutagenesis technology and high-throughput screening to obtain a strain with high tolerance to osmotic pressure, which achieved a 1, 3-propanediol titer of 87 g/L. Furthermore, the gene expression elements suitable for Klebsiella pneumoniae were screened, and metabolic engineering was employed to block redundant metabolic pathways (deletion of ldhA, budA, and aldA) and enhance the synthesis pathway (overexpression of dhaB and yqhD). The titer of 1, 3-propanediol produced by the engineered strain increased to 107 g/L. Finally, in a 5 L fermenter, the optimal strain KP-FMME-6 achieved a 1, 3-propanediol titer of 118 g/L, with a glycerol conversion rate of 42% and productivity of 2.46 g/(h·L), after optimization of the fermentation parameters. This study provides a reference for the industrial production of 1, 3-propanediol.
Assuntos
Fermentação , Klebsiella pneumoniae , Engenharia Metabólica , Propilenoglicóis , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Propilenoglicóis/metabolismo , Engenharia Metabólica/métodos , Glicerol/metabolismo , Mutagênese , Pressão OsmóticaRESUMO
BACKGROUND: Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC). RESULTS: In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out. CONCLUSIONS: Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.
Assuntos
Cisteína , Etanol , Fermentação , Glicerol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vinho , Etanol/metabolismo , Vinho/análise , Glicerol/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cisteína/metabolismo , Evolução Molecular Direcionada , Mutação , Ácido Acético/metabolismoRESUMO
Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Glicerol , Hidroliases , Klebsiella pneumoniae , Propilenoglicóis , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/metabolismo , Hidroliases/genética , Hidroliases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicerol/metabolismo , Propilenoglicóis/metabolismo , Regiões Promotoras Genéticas , FermentaçãoRESUMO
OBJECTIVE: Time-restricted eating (TRE), a dietary approach that confines food intake to specific time windows, has shown metabolic benefits. However, its impact on body weight loss remains inconclusive. The objective of this study was to investigate the influence of early TRE (eTRE) and delayed TRE (dTRE) on fat mobilization using human adipose tissue (AT) cultures. METHODS: Subcutaneous AT was collected from 21 participants with severe obesity. We assessed fat mobilization by measuring glycerol release in AT culture across four treatment conditions: control, eTRE, dTRE, and 24-h fasting. RESULTS: TRE had a significant impact on lipolysis (glycerol release [mean (SD)] in micromoles per hour per gram: control, 0.05 [0.003]; eTRE, 0.10 [0.006]; dTRE, 0.08 [0.005]; and fasting, 0.17 [0.008]; p < 0.0001). Both eTRE and dTRE increased lipolysis compared with the control group, with eTRE showing higher glycerol mobilization than dTRE during the overall 24-h time window, especially at the nighttime/habitual sleep episode (p < 0.0001). Further analysis of TRE based on fasting duration revealed that, independently of the time window, glycerol release increased with fasting duration (in micromoles per hour per gram: 8 h = 0.08 [0.001]; 12 h = 0.09 [0.008]; and 16 h of fasting = 0.12 [0.011]; p < 0.0001). CONCLUSIONS: This study provides insights into the potential benefits of TRE on fat mobilization and may guide the design of future dietary strategies for weight management and metabolic health.
Assuntos
Jejum , Glicerol , Lipólise , Humanos , Glicerol/metabolismo , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Tecido Adiposo/metabolismo , Redução de Peso/fisiologia , Fatores de Tempo , Obesidade Mórbida/dietoterapia , Obesidade Mórbida/metabolismo , Mobilização LipídicaRESUMO
Anoplophora glabripennis is a critical global quarantine pest. Recently, its distribution has been extended to colder and higher-latitude regions. The adaptation to low temperatures is vital for the successful colonization of insects in new environments. However, the metabolic pathways of A. glabripennis larvae under cold stress remain undefined. This study analyzed the larval hemolymph under different low-temperature treatments using LC-MS/MS. The results showed that differential metabolites associated with sugar and lipid metabolism are pivotal in the larval chill coma process. Under low-temperature treatments, the glycerol content increased significantly compared with the control group. Cold stress significantly induced the expression of AglaGK2 and AglaGPDHs. After undergoing RNAi treatment for 48 h, larvae exposed to -20 °C for 1 h showed reduced recovery when injected with ds-AglaGK2 and ds-AglaGPDH1 compared to the control group, indicating that glycerol biosynthesis plays a role in the low-temperature adaptation of A. glabripennis larvae. Our results provide a theoretical basis for clarifying the molecular mechanism of A. glabripennis larvae in response to environmental stresses.
Assuntos
Temperatura Baixa , Glicerol , Proteínas de Insetos , Larva , Animais , Larva/metabolismo , Larva/crescimento & desenvolvimento , Glicerol/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Adaptação Fisiológica , Espectrometria de Massas em Tandem , Hemolinfa/metabolismo , Hemolinfa/química , BesourosRESUMO
Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts.
Assuntos
Peixes-Gato , Sobrevivência Celular , Criopreservação , Espermatogônias , Animais , Masculino , Criopreservação/métodos , Criopreservação/veterinária , Espermatogônias/citologia , Crioprotetores/farmacologia , Testículo/citologia , Dimetil Sulfóxido/farmacologia , Acetamidas/farmacologia , Acetamidas/química , Etilenoglicol/farmacologia , RNA Helicases DEAD-box/metabolismo , Glicerol/farmacologia , Glicerol/metabolismo , Fosfatase Alcalina/metabolismo , Propilenoglicol/farmacologia , Separação Celular/métodosRESUMO
The Saccharomyces cerevisiae DOG genes, DOG1 and DOG2, encode for 2-deoxyglucose-6-phosphate phosphatases. These enzymes of the haloacid dehalogenase superfamily are known to utilize the non-natural 2-deoxyglucose-6-phosphate as their substrate. However, their physiological substrate and hence their biological role remain elusive. In this study, we investigated their potential role as enzymes in biosynthesizing glycerol through an alternative pathway, which involves the dephosphorylation of dihydroxyacetone phosphate into dihydroxyacetone, as opposed to the classical pathway which utilizes glycerol 3-phosphate. Overexpression of DOG1 or DOG2 rescued the osmotic and ionic stress-sensitive phenotype of gpp1∆ gpp2∆ or gpd1∆ gpd2∆ mutants, both affected in the production of glycerol. While small amounts of glycerol were observed in the DOG overexpression strains in the gpp1∆ gpp2∆ background, no glycerol was detected in the gpd1∆ gpd2∆ mutant background. This indicates that overexpression of the DOG enzymes can rescue the osmosensitive phenotype of the gpd1∆ gpd2∆ mutant independent of glycerol production. We also did not observe a drop in glycerol levels in the gpp1∆ gpp2∆ dog1∆ dog2∆ as compared to the gpp1∆ gpp2∆ mutant, indicating that the Dog enzymes are not involved in glycerol biosynthesis. This indicates that Dog enzymes have a distinct substrate and their function within the cell remains undiscovered. IMPORTANCE: Yeast stress tolerance is an important characteristic that is studied widely, not only regarding its fundamental insights but also for its applications within the biotechnological industry. Here, we investigated the function of two phosphatase encoding genes, DOG1 and DOG2, which are induced as part of the general stress response pathway, but their natural substrate in the cells remains unclear. They are known to dephosphorylate the non-natural substrate 2-deoxyglucose-6-phosphate. Here, we show that overexpression of these genes overcomes the osmosensitive phenotype of mutants that are unable to produce glycerol. However, in these overexpression strains, very little glycerol is produced indicating that the Dog enzymes do not seem to be involved in a previously predicted alternative pathway for glycerol production. Our work shows that overexpression of the DOG genes may improve osmotic and ionic stress tolerance in yeast.