RESUMO
Cadaverine is an endogenous metabolite produced by the gut microbiome with various activity in physiological and pathological conditions. However, whether cadaverine regulates pain or itch remains unclear. In this study, we first found that cadaverine may bind to histamine 4 receptor (H4R) with higher docking energy score using molecular docking simulations, suggesting cadaverine may act as an endogenous ligand for H4R. We subsequently found intradermal injection of cadaverine into the nape or cheek of mice induces a dose-dependent scratching response in mice, which was suppressed by a selective H4R antagonist JNJ-7777120, transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine and PLC inhibitor U73122, but not H1R antagonist or TRPA1 antagonist or TRPV4 antagonist. Consistently, cadaverine-induced itch was abolished in Trpv1-/- but not Trpa1-/- mice. Pharmacological analysis indicated that mast cells and opioid receptors were also involved in cadaverine-induced itch in mice. scRNA-Seq data analysis showed that H4R and TRPV1 are mainly co-expressed on NP2, NP3 and PEP1 DRG neurons. Calcium imaging analysis showed that cadaverine perfusion enhanced calcium influx in the dissociated dorsal root ganglion (DRG) neurons, which was suppressed by JNJ-7777120 and capsazepine, as well as in the DRG neurons from Trpv1-/- mice. Patch-clamp recordings found that cadaverine perfusion significantly increased the excitability of small diameter DRG neurons, and JNJ-7777120 abolished this effect, indicating involvement of H4R. Together, these results provide evidences that cadaverine is a novel endogenous pruritogens, which activates H4R/TRPV1 signaling pathways in the primary sensory neurons.
Assuntos
Cadaverina , Gânglios Espinais , Camundongos Endogâmicos C57BL , Prurido , Canais de Cátion TRPV , Animais , Prurido/metabolismo , Prurido/induzido quimicamente , Canais de Cátion TRPV/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Masculino , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Cadaverina/metabolismo , Camundongos , Camundongos Knockout , Humanos , Mastócitos/metabolismo , Mastócitos/efeitos dos fármacos , Canal de Cátion TRPA1/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Capsaicina/análogos & derivadosRESUMO
Migrasomes, the newly discovered cellular organelles that form large vesicle-like structures on the retraction fibers of migrating cells, are thought to be involved in communication between neighboring cells, cellular content transfer, unwanted material shedding, and information integration. Although their formation has been described previously, the molecular mechanisms of migrasome biogenesis are largely unknown. Here, we developed a cell line that overexpresses GFP-tetraspanin4, enabling observation of migrasomes. To identify compounds that regulate migrasome activity in retinal pigment epithelial (RPE) cells, we screened a fecal chemical library and identified cadaverine, a biogenic amine, as a potent migrasome formation inducer. Compared with normal migrating cells, those treated with cadaverine had significantly more migrasomes. Putrescine, another biogenic amine, also increased migrasome formation. Trace amine-associated receptor 8 (TAAR8) depletion inhibited migrasome increase in cadaverine-treated RPE cells, and cadaverine also inhibited protein kinase A phosphorylation. In RPE cells, cadaverine triggers migrasome formation via a TAAR8-mediated protein kinase A signaling pathway.
RESUMO
Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.
Assuntos
Biomarcadores , Cadaverina , Lisina , Periodontite , Humanos , Periodontite/metabolismo , Periodontite/diagnóstico , Cadaverina/metabolismo , Cadaverina/análise , Biomarcadores/metabolismo , Biomarcadores/análise , Lisina/análogos & derivados , Lisina/análise , Lisina/metabolismo , Adulto , Masculino , Fumantes , Feminino , Pessoa de Meia-Idade , Fumar , Saliva/química , Saliva/metabolismoRESUMO
Biogenic amines, produced by bacterial enzymatic reactions in food storage or processing, serve as indicators in food processing industries to assess food quality and freshness. Biogenic amines also often associated with various health problems, including abnormal immune responses and gastrointestinal disease. Previously, salphen base complexes have been reported but still exhibited low fluorescence enhancement upon biogenic amines. This research focused on synthesizing and characterizing new Zn(II) Schiff base complex with indole sidechain to enhance the fluorescence property and exploring their binding behaviour with the biogenic amines, which were phenylethylamine and cadaverine. The Zn(II) indole Schiff base complex's structure was verified by diverse spectroscopic techniques. Then, the binding behaviours between the Zn(II) indole Schiff base complex with the biogenic amines were analyzed using UV-Vis, fluorescence spectroscopy, and Job's plot analysis. UV-Vis binding study results indicated that the synthesized complexes could bind stronger with phenylethylamine than cadaverine, with binding constant, Kb= (8.21 ± 0.58) × 104 M- 1 and (2.506 ± 0.004) × 104 M- 1 respectively. Moreover, Zn(II) indole Schiff base complex-phenylethylamine binding also generated higher fluorescence enhancement than cadaverine, which were 54% and 51% respectively. Based on Job's plot analysis, the complex and biogenic amines were bound in the ratio of 1:1. To conclude, the synthesized complex has promising potential as a sensing material for biogenic amines detection in food. The complex is recommended to be deployed in the development of solid-state fluorescence sensor for biogenic amines detection for monitoring the food spoilage in the food industry in the future.
RESUMO
The microbiome is capable of modulating the bioavailability of chemotherapy drugs, mainly due to metabolizing these agents. Multiple cytostatic bacterial metabolites were recently identified that have cytostatic effects on cancer cells. In this study, we addressed the question of whether a set of cytostatic bacterial metabolites (cadaverine, indolepropionic acid and indoxylsulfate) can interfere with the cytostatic effects of the chemotherapy agents used in the management of breast cancer (doxorubicin, gemcitabine, irinotecan, methotrexate, rucaparib, 5-fluorouracil and paclitaxel). The chemotherapy drugs were applied in a wide concentration range to which a bacterial metabolite was added in a concentration within its serum reference range, and the effects on cell proliferation were assessed. There was no interference between gemcitabine, irinotecan, methotrexate or rucaparib and the bacterial metabolites. Nevertheless, cadaverine and indolepropionic acid modulated the Hill coefficient of the inhibitory curve of doxorubicin and 5-fluorouracil. Changes to the Hill coefficient implicate alterations to the kinetics of the binding of the chemotherapy agents to their targets. These effects have an unpredictable significance from the clinical or pharmacological perspective. Importantly, indolepropionic acid decreased the IC50 value of paclitaxel, which is a potentially advantageous combination.
Assuntos
Neoplasias da Mama , Proliferação de Células , Citostáticos , Doxorrubicina , Fluoruracila , Paclitaxel , Paclitaxel/farmacologia , Fluoruracila/farmacologia , Doxorrubicina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Citostáticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Animais , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Antineoplásicos/farmacologia , Indóis/farmacologiaRESUMO
The response patterns of microbial functional genes involved in biogeochemical cycles to cadaver decay is a central topic of recent environmental sciences. However, the response mechanisms and pathways of the functional genes associated with the carbon (C) and nitrogen (N) cycling to cadaveric substances such as cadaverine and putrescine remain unclear. This study explored the variation of functional genes associated with C fixation, C degradation and N cycling and their influencing factors under cadaverine, putrescine and mixed treatments. Our results showed only putrescine significantly increased the alpha diversity of C fixation genes, while reducing the alpha diversity of N cycling genes in sediment. For the C cycling, the mixed treatment significantly decreased the total abundance of reductive acetyl-CoA pathway genes (i.e., acsB and acsE) and lig gene linked to lignin degradation in water, while only significantly increasing the hydroxypropionate-hydroxybutylate cycle (i.e., accA) gene abundance in sediment. For the N cycling, mixed treatment significantly decreased the abundance of the nitrification (i.e., amoB), denitrification (i.e., nirS3) genes in water and the assimilation pathway gene (i.e., gdhA) in sediment. Environmental factors (i.e., total carbon and total nitrogen) were all negatively associated with the genes of C and N cycling. Therefore, cadaverine and putrescine exposure may inhibit the pathway in C fixation and N cycling, while promoting C degradation. These findings can offer some new insight for the management of amine pollution caused by animal cadavers.
Assuntos
Carbono , Putrescina , Humanos , Animais , Cadaverina , Água , Rios/química , Sedimentos Geológicos/química , Ciclo do Nitrogênio , NitrogênioRESUMO
Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.
Assuntos
Desidrogenases de Carboidrato , Proteínas de Escherichia coli , Cadaverina/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Escherichia coli/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fosfatos/metabolismo , Proteínas de Escherichia coli/genéticaRESUMO
Contamination of antibiotic resistomes due to animal carcass decay has become a serious environmental concern. However, the relationship between main metabolite compounds of corpse decomposition (i.e., putrescine and cadaverine) and antibiotic resistomes remains unclear. To tackle this issue, the response of antibiotic resistance genes (ARGs) and microbiome in aquatic environment to excess putrescine, cadaverine and a mixture of both based on laboratory simulation experiment was investigated by high-throughput quantitative PCR and amplicon sequencing methods. Our results showed putrescine and cadaverine led to the increasing of TC (total carbon) and TN (total nitrogen) both in water and sediment. Under the exposure of putrescine and cadaverine, the total abundance of mobile genetic elements (MGEs) and most ARGs in water was higher than in sediment. In particular, putrescine and cadaverine caused significantly different decreases in alpha diversity of microbial community in water and sediment compared with the control group. Microbial community structures both in water and sediment were also significantly affected by cadaverine and putrescine. Furthermore, putrescine and cadaverine led to different degrees of increases of high-risk ARGs (like mecA) and opportunistic pathogens (like Delftia) in sediment, promoting the prevalence of antibiotic resistant bacteria. In conclusion, our findings revealed the influences of main metabolites of carcass decay on microbiome and resistomes, providing references for risk assessment and pollution management.
Assuntos
Genes Bacterianos , Putrescina , Animais , Cadaverina , Água , Rios , Multiômica , AntibacterianosRESUMO
Herein, the ability of highly porous colorimetric indicators to sense volatile and biogenic amine vapors in real time is presented. Curcumin-loaded polycaprolactone porous fiber mats are exposed to various concentrations of off-flavor compounds such as the volatile amine trimethylamine, and the biogenic amines cadaverine, putrescine, spermidine, and histamine, in order to investigate their colorimetric response. CIELAB color space analysis demonstrates that the porous fiber mats can detect the amine vapors, showing a distinct color change in the presence of down to 2.1 ppm of trimethylamine and ca. 11.0 ppm of biogenic amines, surpassing the limit of visual perception in just a few seconds. Moreover, the color changes are reversible either spontaneously, in the case of the volatile amines, or in an assisted way, through interactions with an acidic environment, in the case of the biogenic amines, enabling the use of the same indicator several times. Finally, yet importantly, the strong antioxidant activity of the curcumin-loaded fibers is successfully demonstrated through DPPHâ and ABTSâ radical scavenging assays. Through such a detailed study, we prove that the developed porous mats can be successfully established as a reusable smart system in applications where the rapid detection of alkaline vapors and/or the antioxidant activity are essential, such as food packaging, biomedicine, and environmental protection.
Assuntos
Antioxidantes , Curcumina , Colorimetria , Aminas Biogênicas/análise , PolímerosRESUMO
Chlamydomonas reinhardtii (C. reinhardtii) is a single-cell green alga that can be easily genetically manipulated. With its favorable characteristics of rapid growth, low cost, non-toxicity, and the ability for post-translational protein modification, C. reinhardtii has emerged as an attractive option for the biosynthesis of various valuable products. To enhance the expression level of exogenous genes and overcome the silencing of foreign genes by C. reinhardtii, synthetic promoters such as the chimeric promoter AR have been constructed and evaluated. In this study, a synthetic promoter GA was constructed by hybridizing core fragments from the natural promoters of the acyl carrier protein gene (ACP2) and the glutamate dehydrogenase gene (GDH2). The GA promoter exhibited a significant increase (7 times) in expressing GUS, over the AR promoter as positive control. The GA promoter also displayed a strong responsiveness to blue light (BL), where the GUS expression was doubled compared to the white light (WL) condition. The ability of the GA promoter was further tested in the expression of another exogenous cadA gene, responsible for catalyzing the decarboxylation of lysine to produce cadaverine. The cadaverine yield driven by the GA promoter was increased by 1-2 times under WL and 2-3 times under BL as compared to the AR promoter. This study obtained, for the first time, a blue light-responsive GDH2 minimal fragment in C. reinhardtii, which delivered a doubling effect under BL when used alone or in hybrid. Together with the strong GA synthetic promoter, this study offered useful tools of synthetic biology to the algal biotechnology field.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cadaverina/metabolismo , Regiões Promotoras Genéticas , Biotecnologia , LuzRESUMO
Necro-leachate, a liquid released during cadaveric decomposition, is considered the main culprit for impacts on cemetery environments. The biogenic amines cadaverine and putrescine make up part of the composition of necro-leachate and have a certain toxicity to different organisms. Springtails are among the most used bioindicators to assess the impacts of soil contaminants. As there are no data on the acute and chronic toxicity of springtails exposed to cadaverine and putrescine, the objective of this study was to evaluate the toxic potential of both amines, under the behavioral effect of avoidance and reproduction in the species Folsomia candida. Springtails were exposed to soils contaminated with different concentrations of cadaverine and putrescine, and different mixtures of these amines. To evaluate the avoidance and reproduction test, the individuals were exposed for periods of 48 h and 28 days, respectively. The results obtained in the avoidance test showed that springtails exhibited a preference for the treated soil in both isolated and mixed treatments. The chronic evaluation assays showed that the reproduction was affected, particularly in the treatments with combined amines, resulting in a reduction in the total number of juveniles. From the results, it is possible to infer that the methods applied in this research have provided information that will contribute to a better understanding of the toxicity of putrefactive biogenic amines, since there exist few ecotoxicological studies carried out with these amines, and especially with those from cemetery environments.
Assuntos
Artrópodes , Putrescina , Humanos , Animais , Cadaverina , Monitoramento Ambiental , Cadáver , Aminas Biogênicas/toxicidade , SoloRESUMO
Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5'-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.
Assuntos
Proteínas Arqueais , Bactérias , Proteínas de Bactérias , Poliaminas Biogênicas , Carboxiliases , Euryarchaeota , Evolução Molecular , Ornitina Descarboxilase , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Poliaminas Biogênicas/biossíntese , Poliaminas Biogênicas/química , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/metabolismo , Euryarchaeota/enzimologia , Euryarchaeota/genética , Ornitina Descarboxilase/química , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Biotina/biossíntese , Cadaverina/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Transaminases/metabolismo , Acetil-CoA Carboxilase/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Biotinilação , Carbono-Nitrogênio Ligases/genética , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transaminases/genéticaRESUMO
Salmonella and E. coli synthesize, import, and export cadaverine, putrescine, and spermidine to maintain physiological levels and provide pH homeostasis. Both low and high intracellular levels of polyamines confer pleiotropic phenotypes or lethality. Here, we demonstrate that the previously uncharacterized inner membrane protein PaeA (YtfL) is required for reducing cytoplasmic cadaverine and putrescine concentrations. We identified paeA as a gene involved in stationary phase survival when cells were initially grown in acidic medium, in which they produce cadaverine. The paeA mutant is also sensitive to putrescine, but not to spermidine or spermine. Sensitivity to external cadaverine in stationary phase is only observed at pH > 8, suggesting that the polyamines need to be deprotonated to passively diffuse into the cell cytoplasm. In the absence of PaeA, intracellular polyamine levels increase and the cells lose viability. Degradation or modification of the polyamines is not relevant. Ectopic expression of the known cadaverine exporter, CadB, in stationary phase partially suppresses the paeA phenotype, and overexpression of PaeA in exponential phase partially complements a cadB mutant grown in acidic medium. These data support the hypothesis that PaeA is a cadaverine/putrescine exporter, reducing potentially toxic levels under certain stress conditions.
Assuntos
Cadaverina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Putrescina/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Espermidina/metabolismoRESUMO
1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.
Assuntos
Escherichia coli , Engenharia Metabólica , Cadaverina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/genética , Glucose/metabolismoRESUMO
BACKGROUND: Polyamide (nylon) is an important material, which has aroused plenty of attention from all aspects. PA 5.4 is one kind of nylon with excellent property, which consists of cadaverine and succinic acid. Due to the environmental pollution, bio-production of cadaverine and succinic acid has been more attractive due to the less pollution and environmental friendliness. Microbes, like Escherichia coli, has been employed as cell factory to produce cadaverine and succinic acid. However, the accumulation of cadaverine will cause severe damage on cells resulting in inhibition on cell growth and cadaverine production. Herein, a novel two stage co-production of succinic acid and cadaverine was designed based on an efficient thermos-regulated switch to avoid the inhibitory brought by cadaverine. RESULTS: The fermentation process was divided into two phase, one for cell growth and lysine production and the other for cadaverine and succinic acid synthesis. The genes of ldhA and ackA were deleted to construct succinic acid pathway in cadaverine producer strain. Then, a thermal switch system based on pR/pL promoter and CI857 was established and optimized. The fermentation conditions were investigated that the optimal temperature for the first stage was determined as 33 â and the optimal temperature for the second stage was 39 â. Additionally, the time to shifting temperature was identified as the fermentation anaphase. For further enhance cadaverine and succinic acid production, a scale-up fermentation in 5 L bioreactor was operated. As a result, the titer, yield and productivity of cadaverine was 55.58 g/L, 0.38 g/g glucose and 1.74 g/(L·h), respectively. 28.39 g/L of succinic acid was also obtained with yield of 0.19 g/g glucose. CONCLUSION: The succinic acid metabolic pathway was constructed into cadaverine producer strain to realize the co-production of succinic acid and cadaverine. This study provided a novel craft for industrial co-production of cadaverine and succinic acid.
Assuntos
Escherichia coli , Ácido Succínico , Cadaverina , Escherichia coli/genética , Nylons , GlucoseRESUMO
OBJECTIVES: 1,5-pentanediamine (cadaverine) is a C5 platform chemical, also an important raw material for bio-polyamide PA5X. With increasing concerns about the depletion of fossil resources and global environmental protection, cadaverine bio-production has attracted more attentions. RESULTS: Here, a microbial consortium consisting of Corynebacterium glutamicum cgl-FDK and Escherichia coli BL-ABST-Spy was constructed to de novo synthesize cadaverine utilizing glycerol as the sole carbon resource. The glycerol utilization pathway was initially constructed in C. glutamicum cgl-FDK to produce lysine from glycerol. Then, the pyridoxal 5'-phosphate (PLP) biosynthesis pathway and SpyTag/SpyCatcher protein-ligation system for lysine decarboxylase (CadA) and cadaverine-lysine antiporter protein (CadB) were introduced into E. coli BL-ABST-Spy to synthesize cadaverine from lysine. Furthermore, the fermentation conditions of microbial consortium were optimized and the cadaverine production reached 9.3 g/L with glycerol as the sole carbon source. CONCLUSIONS: This work provides a promising strategy for efficiently producing cadaverine from glycerol with an artificial microbial consortium.
Assuntos
Corynebacterium glutamicum , Glicerol , Cadaverina , Glicerol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lisina/metabolismo , Consórcios Microbianos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Fosfato de Piridoxal/metabolismo , Carbono/metabolismoRESUMO
Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.
Assuntos
Trifosfato de Adenosina/metabolismo , Cadaverina/química , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , Saccharomyces cerevisiae , Ágar/química , Biotecnologia/métodos , Biotransformação , Cadaverina/metabolismo , Carboxiliases , Fermentação , Microbiologia Industrial/instrumentação , Microbiologia Industrial/métodos , Lisina/química , Lisina/metabolismo , Polímeros/química , Piridoxal , RegeneraçãoRESUMO
Micro-cantilever sensors are a known reliable tool for gas sensing in industrial applications. We have demonstrated the application of cantilever sensors on the detection of a meat freshness volatile biomarker (cadaverine), for determination of meat and fish precise expiration dates. For achieving correct target selectivity, the cantilevers need to be functionalized with a cadaverine-selective binder, based on a cyclam-derivative. Cantilever surface properties such as surface energy strongly influence the binder morphology and material clustering and, therefore, target binding. In this paper, we explore how chemical and physical surface treatments influence cantilever surface, binder morphology/clustering and binding capabilities. Sensor measurements with non-controlled surface properties are presented, followed by investigations on the binder morphology versus surface energy and cadaverine capture. We demonstrated a method for hindering binder crystallization on functionalized surfaces, leading to reproducible target capture. The results show that cantilever surface treatment is a promising method for achieving a high degree of functionalization reproducibility for industrial cantilever sensors, by controlling binder morphology and uniformity.
Assuntos
Técnicas Biossensoriais , Animais , Reprodutibilidade dos Testes , Propriedades de SuperfícieRESUMO
The identification and characterization of ligand-receptor binding sites are important for drug development. Trace amine-associated receptors (TAARs, members of the class A GPCR family) can interact with different biogenic amines and their metabolites, but the structural basis for their recognition by the TAARs is not well understood. In this work, we have revealed for the first time a group of conserved motifs (fingerprints) characterizing TAARs and studied the docking of aromatic (ß-phenylethylamine, tyramine) and aliphatic (putrescine and cadaverine) ligands, including gamma-aminobutyric acid, with human TAAR1 and TAAR6 receptors. We have identified orthosteric binding sites for TAAR1 (Asp68, Asp102, Asp284) and TAAR6 (Asp78, Asp112, Asp202). By analyzing the binding results of 7500 structures, we determined that putrescine and cadaverine bind to TAAR1 at one site, Asp68 + Asp102, and to TAAR6 at two sites, Asp78 + Asp112 and Asp112 + Asp202. Tyramine binds to TAAR6 at the same two sites as putrescine and cadaverine and does not bind to TAAR1 at the selected Asp residues. ß-Phenylethylamine and gamma-aminobutyric acid do not bind to the TAAR1 and TAAR6 receptors at the selected Asp residues. The search for ligands targeting allosteric and orthosteric sites of TAARs has excellent pharmaceutical potential.