Combining directed evolution with high cell permeability for high-level cadaverine production in engineered Escherichia coli.

The biosynthesis of cadaverine from lysine is an environmentally promising technology, that could contribute to a more sustainable approach to manufacturing bio-nylon 5X. However, the titer of biosynthesized cadaverine has still not reached a sufficient level for industrial production. A powerful green cell factory was developed to enhance cadaverine production by regulating lipopolysaccharide (LPS) genes and improving membrane permeability. Firstly, 10 LPS mutant strains were constructed and the effect on the growth was investigated. Then, the lysine decarboxylase (CadA) was overexpressed in 10 LPS mutant strains of Escherichia coli MG1655 and the ability to produce cadaverine was compared. Using 20.0 g L-1 of L-lysine hydrochloride (L-lysine-HCl) as the substrate for the biotransformation reaction, Cad02 and Cad06 strains exhibited high production levels of cadaverine, with 8.95 g L-1 and 7.55 g L-1 respectively while the control strain Cad00 only 4.92 g L-1 . Directed evolution of CadA was also used to improve its stability under alkaline conditions. The cadaverine production of the Cad02-M mutant stain increased by 1.86 times at pH 8.0. Finally, the production process was scaled up using recombinant whole cells as catalysts, achieving a high titer of 211 g L-1 cadaverine (96.8%) by fed-batch bioconversion. This study demonstrates the potential role of LPS in enhancing the efficiency of mass transfer between substrate and enzymes in vivo by increasing cell permeability. The results indicate that the argumentation of cell permeability could not only significantly enhance the biotransformation efficiency of cadaverine, but also provide a universally applicable, straightforward, environment-friendly, and cost-effective method for the biosynthesis of other high-value chemicals.

Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (ERLIC) for the Quantitative Analysis of Polyamines.

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules.

Cadaverine and putrescine exposure influence carbon and nitrogen cycling genes in water and sediment of the Yellow River.

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.

Engineering RuBisCO-based shunt for improved cadaverine production in Escherichia coli.

The process of biological fermentation is often accompanied by the release of CO2, resulting in low yield and environmental pollution. Refixing CO2 to the product synthesis pathway is an attractive approach to improve the product yield. Cadaverine is an important diamine used for the synthesis of bio-based polyurethane or polyamide. Here, aiming to increase its final production, a RuBisCO-based shunt consisting of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulate kinase (PRK) was expressed in cadaverine-producing E. coli. This shunt was calculated capable of increasing the maximum theoretical cadaverine yield based on flux model analysis. When a functional RuBisCO-based shunt was established and optimized in E. coli, the cadaverine production and yield of the final engineered strain reached the highest level, which were 84.1 g/L and 0.37 g/g Glucose, respectively. Thus, the design of in situ CO2 fixation provides a green and efficient industrial production process.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BACKGROUND: BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. OBJECTIVES: To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. METHODS: E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). RESULTS: The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. CONCLUSIONS: Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Orchestrating Precision within the Tumor Microenvironment by Biomimetic Nanoprodrugs for Effective Tumor Therapy.

Malignant tumors are still one of the most deadly diseases that threaten human life and health. However, developing new drugs is challenging due to lengthy trials, funding constraints, and regulatory approval procedures. Consequently, researchers have devoted themselves to transforming some clinically approved old drugs into antitumor drugs with certain active ingredients, which have become an attractive alternative. Disulfiram (DSF), an antialcohol medication, can rapidly metabolize in the physiological environment into diethyldithiocarbamate (DTC) which can readily react with Cu2+ ions in situ to form the highly toxic bis(N,N-diethyldithiocarbamate)-copper(II) (CuET) complex. In this study, DSF is loaded into mesoporous dopamine nanocarriers and surface-chelated with tannin and Cu2+ to construct M-MDTC nanoprodrugs under the camouflage of K7 tumor cell membranes. After intravenous injection, M-MDTC nanoprodrugs successfully reach the tumor sites with the help of mediated cell membranes. Under slightly acidic pH and photothermal stimulation conditions, DSF and Cu2+ are simultaneously released, forming a highly toxic CuET to kill tumor cells in situ. The generated CuET can also induce immunogenic cell death of tumor cells, increase the proportion of CD86+ CD80+ cells, and promote dendritic cell maturation. In vitro and in vivo studies of M-MDTC nanoprodrugs have shown excellent tumor-cell-killing ability and solid tumor suppression. This approach enables in situ amplification of chemotherapy in the tumor microenvironment, achieving an effective antitumor treatment.

Decarboxylase activity of the non-starter lactic acid bacterium Loigolactobacillus rennini gives crack defects in Gouda cheese through the production of γ-aminobutyric acid.

Ten Gouda cheese wheels with an age of 31 weeks from six different batch productions were affected by a crack defect and displayed an unpleasant off-flavor. To unravel the causes of these defects, the concentrations of free amino acids, other organic acids, volatile organic compounds, and biogenic amines were quantified in zones around the cracks and in zones without cracks, and compared with those of similar Gouda cheeses without crack defect. The Gouda cheeses with cracks had a significantly different metabolome. The production of the non-proteinogenic amino acid γ-aminobutyric acid (GABA) could be unraveled as the key mechanism leading to crack formation, although the production of the biogenic amines cadaverine and putrescine contributed as well. High-throughput amplicon sequencing of the full-length 16S rRNA gene based on whole-community DNA revealed the presence of Loigolactobacillus rennini and Tetragenococcus halophilus as most abundant non-starter lactic acid bacteria in the zones with cracks. Shotgun metagenomic sequencing allowed to obtain a metagenome-assembled genome of both Loil. rennini and T. halophilus. However, only Loil. rennini contained genes necessary for the production of GABA, cadaverine, and putrescine. Metagenetics further revealed the brine and the rennet used during cheese manufacturing as the most plausible inoculation sources of both Loil. rennini and T. halophilus.IMPORTANCECrack defects in Gouda cheeses are still poorly understood, although they can lead to major economic losses in cheese companies. In this study, the bacterial cause of a crack defect in Gouda cheeses was identified, and the pathways involved in the crack formation were unraveled. Moreover, possible contamination sources were identified. The brine bath might be a major source of bacteria with the potential to deteriorate cheese quality, which suggests that cheese producers should regularly investigate the quality and microbial composition of their brines. This study illustrated how a multiphasic approach can understand and mitigate problems in a cheese company.

Mixed-Linkage Donor-Acceptor Covalent Organic Framework as a Turn-On Fluorescent Sensor for Aliphatic Amines.

Amine determination is crucial to our daily life, including the prevention of pollution, the treatment of certain disorders, and the evaluation of food quality. Herein, a mixed-linkage donor-acceptor covalent organic framework (named DSE-COF) was first constructed by the polymerization between 2,4-dihydroxybenzene-1,3,5-tricarbaldehyde (DTA) and 4,4'-(benzo[c][1,2,5]selenadiazole-4,7-diyl)dianiline (SEZ). DSE-COF displayed superior turn-on fluorescent responses to primary, secondary, and tertiary aliphatic amines, such as cadaverine, isopropylamine, sec-butylamine, cyclohexylamine, hexamethylenediamine, di-n-butylamine, and triethylamine in absolute acetonitrile than other organic species. Further experiments and theoretical calculations demonstrated that the combination of intramolecular charge transfer (ICT) and photoinduced electron transfer (PET) effects between the DSE-COF and aliphatic amines resulted in enhanced fluorescence. Credibly, DSE-COF can quantitatively detect cadaverine content in actual pork samples with satisfactory results. In addition, DSE-COF-based test papers could rapidly monitor cadaverine from real pork samples, manifesting the potential application of COFs in food quality inspection.

Evaluation of the ecotoxicological effects of biogenic amines derived from cadaverous putrefaction on springtails Folsomia candida.

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.

A novel biochemistry approach combined with MALDI-TOF MS to discriminate Escherichia coli and Shigella species.

Escherichia coli and Shigella spp. are closely related, making it crucial to accurately identify them for disease control and prevention. In this study, we utilized MALDI-TOF MS to identify characteristic peaks of decarboxylation products of lysine and ornithine to distinguish between E. coli and Shigella spp. Our findings indicate that the peak at m/z 103.12 ± 0.1 of the product cadaverine from lysine decarboxylase is unique to E. coli, while all Shigella species lack the m/z 103.12 ± 0.1 peak. However, S. sonnei and S. boydii serotype C13 exhibit a specific peak at m/z 89.10 ± 0.1, which is the product of putrescine from ornithine decarboxylase. We were able to correctly identify 97.06% (132 of 136) of E. coli and Shigella isolates and 100% (8 of 8) of S. sonnei isolates using this biochemical-based MALDI-TOF MS detection system. This technology is advantageous for its high-throughput, high quality, and ease of operation, and is of significant value for the diagnosis of E. coli and Shigella-related diseases.

Alleviation role of exogenous cadaverine on cell cycle, endogenous polyamines amounts and biochemical enzyme changes in barley seedlings under drought stress.

Cadaverine (Cad), which has an independent synthesis pathway compared to other polyamine (PA) types, contributes to the health of plants by regulating plant growth and development, abiotic stress tolerance and antioxidant defense mechanisms. In this work, experiments were carried out to understand the effects of exogenous Cad (10 µM) application under drought stress (%22 PEG 6000) and without stress on cell cycle, total protein content, endogenous PA levels, and biochemical enzyme activities in barley (Hordeum vulgare cv. Burakbey) considering the potential of Cad to stimulate the drought-related tolerance system. Cad application in a stress-free environment showed an effect almost like low-impact drought stress, causing changes in all parameters examined compared to samples grown in distilled water environment (Control). The results clearly show that Cad applied against the negative effects of drought stress on all parameters creates a drought resistance mechanism of the plant. Accordingly, Cad applied together with drought stress increased the density of cells in the cell cycle (G1-S and S-G2 phases) and the amount of endogenous (spermidine 10% and spermine 40%) PAs. In addition, while superoxide dismutase (SOD) (5%), (CAT) (55%) and ascorbate peroxidase (APX) (18%) enzyme levels increased, a stress response mechanism occurred due to the decrease in total protein content (20%) and malondialdehyde (MDA) (80%). As a result, exogenous application of 10 µM Cad showed that it reduced the negative effects of drought stress on endogenous PA amounts, cell division and biochemical activities in barley.

Specific Gene Expression in Pseudomonas Putida U Shows New Alternatives for Cadaverine and Putrescine Catabolism.

Pseudomonas putida strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase (spuC1 and spuC2 genes) and a ɣ-aminobutyrate aminotransferase (gabT1 and gabT2 genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, spuC1 is upregulated by putrescine, whereas spuC2 seems to be exclusively induced by cadaverine. However, gabT1 increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although gabT2 does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in P. aeruginosa and Escherichia coli concerning P. putida strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism.

A Blue Light-Responsive Strong Synthetic Promoter Based on Rational Design in Chlamydomonas reinhardtii.

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.

Evaluating biogenic amines and pH levels in farmed rainbow trout (Oncorhynchus mykiss) for freshness assessment during storage.

This study examined the production of eight key biogenic amines (methylamine, tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in 120 samples of farmed rainbow trout during various storage conditions, and determined any accompanying variations in pH. The main objective of this study was to identify which of the eight biogenic amines could be used as chemical markers to evaluate the quality of farmed rainbow trout. Histamine and tryptamine were not present in any of the samples, and the levels of cadaverine were inconsistent. The levels of putrescine significantly increased at 0 °C (by day 9), 2 °C (by day 8), and 4 °C (by day 4). Tyramine, spermidine, and spermine levels exhibited fluctuations but had a significant positive correlation with the levels of putrescine. The pH levels slightly increased from their initial values across all storage temperatures, with no significant variations observed. Based on the results, it can be concluded that putrescine may serve as an effective marker of the freshness of farmed rainbow trout during storage.

Aldehyde-functionalized cellulose as reactive sorbents for the capture and retention of polyamine odor molecules associated with chronic wounds.

Aldehyde-functionalized cellulose (AFC) was prepared by oxidizing cellulose with sodium metaperiodate. The reaction was characterized by Schiff's test, FT-IR, and UV-vis study. AFC was evaluated as a reactive sorbent for controlling polyamine-based odor from chronic wounds, and its performance was compared with charcoal, one of the most widely utilized odor-control sorbents through physisorption. Cadaverine was used as the model odor molecule. A liquid chromatography/mass spectrometry (LC/MS) method was established to quantify the compound. AFC was found to rapidly react with cadaverine through the Schiff-base reaction, which was confirmed by FT-IR, visual observation, CHN elemental analysis, and the ninhydrin test. The sorption and desorption behaviors of cadaverine onto AFC were quantified. With clinic-relevant cadaverine concentrations, AFC demonstrated much better sorption performance than charcoal. At even higher cadaverine concentrations charcoal showed higher sorption capacity, probably due to its high surface area. On the other hand, in desorption studies, AFC retained much more of the sorbed cadaverine than charcoal. When AFC and charcoal were combined, the pair demonstrated excellent sorption and desorption behaviors. The XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay confirmed that AFC has very good in vitro biocompatibility. These results suggest that AFC-based reactive sorption can be a new strategy to control odors associated with chronic wounds for improved healthcare.

Ultrasensitive Gas Detection Based on Electrically Enhanced Nanoplasmonic Sensor with Graphene-Encased Gold Nanorod.

Nanoplasmonic sensors are a widely known concept and have been studied with various applications. Among them, gas detection is engaging attention in many fields. However, the analysis performance of nanoplasmonic sensors based on refractive index confined to the metal nanostructure characteristics causes challenges in gas detection. In this study, we develop a graphene-encased gold nanorod (AuNR)-based nanoplasmonic sensor to detect cadaverine gas. The graphene-encased AuNR (Gr@AuNR) presents an ultrasensitive peak wavelength shift even with tiny molecules. In addition, the external potential transmitted through graphene induces an additional shift. A chemical receptor is immobilized on Gr@AuNR (CR@Gr@AuNR) for selectively capturing cadaverine. The CR@Gr@AuNR achieves ultrasensitive detection of cadaverine gas, and the detection limit is increased to 15.99 ppb by applying a voltage to graphene. Furthermore, the experimental results of measuring cadaverine generated from spoiled pork show the practicality of CR@Gr@AuNR. The strategy of external-boosted nanoplasmonics provides new insight into plasmonic sensing and applications.

The Interplay of AphB and CadC to Activate Acid Resistance of Vibrio campbellii.

Bacteria have evolved different systems to sense and adapt to acid stress. For example, Vibrio campbellii, a marine pathogen for invertebrates, encounters acidic conditions in the digestive glands of shrimp. The main acid resistance system of V. campbellii is the Cad system, which is activated when cells are in a low-pH, amino acid-rich environment. The Cad system consists of the pH-responsive transcriptional activator CadC, the lysine decarboxylase CadA, and the lysine/cadaverine antiporter CadB. In many Vibrio species, the LysR-type transcriptional regulator AphB is involved in the regulation of the Cad system, but its precise role is unclear. Here, we examined AphB of V. campbellii in vivo and in vitro in the context of Cad activation. At low pH, an aphB deletion mutant was less able to grow and survive compared with the wild-type because it did not excrete sufficient alkaline cadaverine to increase the extracellular pH. AphB was found to upregulate the transcription of cadC, thereby increasing its protein copy number per cell. Moreover, AphB itself was shown to be a pH-sensor, and binding to the cadC promoter increased under low pH, as shown by surface plasmon resonance spectroscopy. By monitoring the activation of the Cad system over a wide range of pH values, we found that AphB-mediated upregulation of cadC not only adjusts CadC copy numbers depending on acid stress strength, but also affects the response of individual cells and thus the degree of heterogeneous Cad system activation in the V. campbellii population. IMPORTANCE Acid resistance is an important property not only for neutralophilic enteric bacteria such as Escherichia, Yersinia, and Salmonella, but also for Vibrio. To counteract acidic threats, the marine Vibrio campbellii, a pathogen for various invertebrates, activates the acid-resistance Cad system. The transcriptional activator of the Cad system is CadC, an extracellular pH-sensor. The expression of cadC is upregulated by the transcriptional regulator AphB to achieve maximum expression of the components of the Cad system. In vitro studies demonstrate that AphB binds more tightly to the DNA under low pH. The interplay of two pH-responsive transcriptional activators allows tight control of the activity of the Cad system.

Evaluation of the stability of polyamines in human serum at different storage temperatures using capillary electrophoresis with indirect ultraviolet detection.

Polyamines are low molecular weight compounds that are present in all living organisms. They are related to the pathological processes, and have been studied as biomarkers for tumor progression, being analyzed in patients' biological fluids. However, polyamines can undergo degradation in serum samples, depending on storage conditions, which impairs their quantification in these matrices. In this work, capillary electrophoresis using indirect ultraviolet detection has been developed and applied to evaluate the stability of polyamines [cadaverine (Cad), putrescine (Put), spermine (Spm), and spermidine (Spd)] in human serum at different storage temperatures. By using this method, Cad, Put, Spm, and Spd were separated in less than 4 min. The range of the correlation coefficients was 0.993-0.998. The corresponding limits of detection and quantification were as follows (in mg L-1 ): Spm: 0.209 and 0.697; Spd: 0.165 and 0.549; Put: 0.189 and 0.632; Cad: 0.125 and 0.417. Besides, the coefficient of variation was lower than 1% for all analytes and the recovery was 92%-110%. The method was successfully applied for polyamines spiked in human serum samples from healthy people. The results showed that the degradation of polyamines was lower in samples stored in a freezer (-20°C).

Investigation of enzymatic quality and quantity using pyridoxal 5'-phosphate (PLP) regeneration system as a decoy in Escherichia coli.

Pyridoxal 5'-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

Dysbiosis of Oral Microbiota and Metabolite Profiles Associated with Type 2 Diabetes Mellitus.

Several previous studies have shown that oral microbial disorders may be closely related to the occurrence and development of type 2 diabetes mellitus (T2DM). However, whether the function of oral microorganisms and their metabolites have changed in patients with T2DM who have not suffered from any oral diseases has not been reported. We performed metagenomic analyses and nontargeted metabolic analysis of saliva and supragingival plaque samples from patients with T2DM who have not suffered any oral diseases and normal controls. We found that periodontal pathogens such as Porphyromonas gingivalis and Prevotella melaninogenica were significantly enriched, while the abundances of dental caries pathogens such as Streptococcus mutans and Streptococcus sobrinus were not significantly different in patients with T2DM compared to those in normal controls. Metabolomic analyses showed that the salivary levels of cadaverine and L-(+)-leucine of patients with T2DM were significantly higher than those of normal controls, while the supragingival plaque levels of N-acetyldopamine and 3,4-dimethylbenzoic acid in patients with T2DM were significantly higher than those in the normal controls. Additionally, we identified the types of oral microorganisms related to the changes in the levels of circulating metabolites, and the oral microorganisms were involved in the dysregulation of harmful metabolites such as cadaverine and n, n-dimethylarginine. Overall, our study first described the changes in the composition of oral microorganisms and their metabolites in patients with T2DM who have not suffered any oral diseases, which will provide a direct basis for finding oral biomarkers for early warning of oral diseases in T2DM. IMPORTANCE The incidence of oral diseases in type 2 diabetic patients might increase, and the severity might also be more serious. At present, the relationship between oral microorganisms and type 2 diabetes mellitus (T2DM) has become a hot topic in systemic health research. However, whether the function of oral microorganisms and their metabolites have changed in patients with T2DM who have not suffered from any oral diseases has not been reported. We found that even if the oral condition of T2DM is healthy, their oral microbes and metabolites have changed, thus increasing the risk of periodontal disease. Our study first described the changes in the composition of oral microorganisms and their metabolites in T2DM who have not suffered any oral diseases and revealed the correlation between oral microorganisms and their metabolites, which will provide a direct basis for finding oral biomarkers for early warning of oral diseases in patients with T2DM.