Characterization of the enzyme kinetics of EMP and HMP pathway in Corynebacterium glutamicum: reference for modeling metabolic networks

The model of intracellular metabolic network based on enzyme kinetics parameters plays an important role in understanding the intracellular metabolic process of Corynebacterium glutamicum, and constructing such a model requires a large number of enzymological parameters. In this work, the genes encoding the relevant enzymes of the EMP and HMP metabolic pathways from Corynebacterium glutamicum ATCC 13032 were cloned, and engineered strains for protein expression with E.coli BL21 and P.pastoris X33 as hosts were constructed. The twelve enzymes (GLK, GPI, TPI, GAPDH, PGK, PMGA, ENO, ZWF, RPI, RPE, TKT, and TAL) were successfully expressed and purified by Ni2+ chelate affinity chromatography in their active forms. In addition, the kinetic parameters (Vmax, Km, and Kcat) of these enzymes were measured and calculated at the same pH and temperature. The kinetic parameters of enzymes associated with EMP and the HMP pathway were determined systematically and completely for the first time in C.glutamicum. These kinetic parameters enable the prediction of key enzymes and rate-limiting steps within the metabolic pathway, and support the construction of a metabolic network model for important metabolic pathways in C.glutamicum. Such analyses and models aid in understanding the metabolic behavior of the organism and can guide the efficient production of high-value chemicals using C.glutamicum as a host

Application of Nanomaterial Modified Aptamer-Based Electrochemical Sensor in Detection of Heavy Metal Ions

Heavy metal pollution resulting from significant heavy metal waste discharge is increasingly serious. Traditional methods for the detection of heavy metal ions have high requirements on external conditions, so developing a sensitive, simple, and reproducible detection method is becoming an urgent need. The aptamer, as a new kind of artificial probe, has received more attention in recent years for its high sensitivity, easy acquisition, wide target range, and wide use in the detection of various harmful substances. The detection platform that an aptamer-based electrochemical biosensor (E-apt sensor) provides is a new approach for the detection of heavy metal ions. Nanomaterials are particularly important in the construction of E-apt sensors, as they can be used as aptamer carriers or sensitizers to stimulate or inhibit electrochemical signals, thus significantly improving the detection sensitivity. This review summarizes the application of different types of nanomaterials in E-apt sensors. The construction methods and research progress of the E-apt sensor based on different working principles are systematically introduced. Moreover, the advantages and challenges of the E-apt sensor in heavy metal ion detection are summarized

Efficient expression of an alkaline pectin lyase from Bacillus licheniformis in Pichia pastoris

Abstract Pectin lyase (PMGL) is an industrially important enzyme with widespread applications in the food, paper, and textile industries, owing to its capacity for direct degradation of highly esterified pectin. In this study, PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P. pastoris. Furthermore, diverse strategies, encompassing the optimization of expression cassette components, elevation of gene dosage, and co-expression of chaperone factors, were employed to augment PMGL-Ba production in P. pastoris. The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements. By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup, a strain yielding high PMGL-Ba production was achieved. In shake flask fermentation lasting 144 h, the total protein concentration reached 1.81 g/L, and the enzyme activity reached 1821.36 U/mL. For further scale up production, high-density fermentation transpired in a 5 L fermenter for 72 h. Remarkably, the total protein concentration increased to 12.49 g/L, and the enzyme activity reached an impressive 12668.12 U/mL. The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications

Correction: Xie et al. Selection and Application of ssDNA Aptamers for Fluorescence Biosensing Detection of Malachite Green. Foods 2022, 11, 801

In the original publication [...

Acute and Chronic Toxicity of Binary Mixtures of Bisphenol A and Heavy Metals

Bisphenol A (BPA) and heavy metals are widespread contaminants in the environment. However, the combined toxicities of these contaminants are still unknown. In this study, the bioluminescent bacteria Vibrio qinghaiensis Q67 was used to detect the single and combined toxicities of BPA and heavy metals, then the joint effects of these contaminants were evaluated. The results show that chronic toxicities of chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), nickel (Ni), and BPA were time–dependent; in fact, the acute toxicities of these contaminants were stronger than the chronic toxicities. Furthermore, the combined toxicities of BPA and heavy metals displayed BPA + Hg > BPA + Cr > BPA + As > BPA + Ni > BPA + Pb > BPA + Cd in the acute test and BPA + Hg > BPA + Cd > BPA + As > BPA + Cd in the chronic test, which suggested that the combined toxicity of BPA and Hg was stronger than that of other mixtures in acute as well as chronic tests. Additionally, both CA and IA models underestimated the toxicities of mixtures at low concentrations but overestimated them at high concentrations, which indicates that CA and IA models were not suitable to predict the toxicities of mixtures of BPA and heavy metals. Moreover, the joint effects of BPA and heavy metals mainly showed antagonism and additive in the context of acute exposure but synergism and additive in the context of chronic exposure. Indeed, the difference in the joint effects on acute and chronic exposure can be explained by the possibility that mixtures inhibited cell growth and luminescence in chronic cultivation. The chronic toxicity of the mixture should be considered if the mixture results in the inhibition of the growth of cells

Prediction of prognosis and immunotherapy response in lung adenocarcinoma based on CD79A, DKK1 and VEGFC

Background: Tumor immune microenvironment (TIME) is crucial for tumor initiation, progression, and metastasis; however, its relationship with lung adenocarcinoma (LUAD) is unknown. Traditional predictive models screen for biomarkers that are too general and infrequently associated with immune genes. Methods: RNA sequencing data of LUAD patients and immune-related gene sets were retrieved from public databases. Using the common genes shared by The Cancer Genome Atlas (TCGA) and Immunology Database and Analysis Portal (ImmPort), differential gene expression analysis, survival analysis, Lasso regression analysis, and univariate and multivariate Cox regression analyses were performed to generate a novel risk score model. LUAD cohort in International Cancer Genome Consortium (ICGC), GSE68465 cohort in Gene Expression Omnibus (GEO) and an immunohistochemical assay were used to validate the key genes constructed risk score. The LUAD-related prognosis, clinical indicators, immune infiltrate characteristics, response to immunotherapy, and response to chemotherapeutic agents in different risk groups were evaluated by CIBERSORT, ImmuCellAI, pRRophetic and other tools. Results: The risk score model was constructed using CD79a molecule (CD79A), Dickkopf WNT signaling pathway inhibitor 1 (DKK1), and vascular endothelial growth factor C (VEGFC). High risk score was identified as a negative predictor for overall survival (OS) in subgroup analyses with tumor stage, TNM classification, therapy outcome, and ESTIMATE scores (P < 0.05). Low risk score was positively associated with plasma cells, memory B cells, CD8 T cells, regulatory T cells and γδT cells (P < 0.05). In low-risk group, programmed cell death 1 receptor (PD1), cytotoxic T-lymphocyte associated protein 4 (CTLA4), and lymphocyte activating 3 (LAG3) and indoleamine 2,3-dioxygenase (IDO) were more robustly expressed (P < 0.05). The treatment responses of immune checkpoint blockade (ICB) therapy and chemotherapy were likewise superior in low-risk group (P < 0.05). In immunohistochemical analysis, the tumor group had significantly higher levels of CD79A, DKK1, and VEGFC than the adjacent normal group (P < 0.01). Conclusions: CD79A, DKK1 and VEGFC are important differential genes related to LUAD, risk score could reliably predict prognosis, composition of TIME and immunotherapy responses in LUAD patients. The excellent performance of the risk model shows its strong and broad application potential

Additional file 1 of Efficient expression of an alkaline pectin lyase from Bacillus licheniformis in Pichia pastoris

Supplementary Material