Applied Microbiology and Biotechnology最新文献

Cysteine cathepsins such as cathepsin B and L play an important role in numerous diseases like acute pancreatitis or SARS-CoV-2 and therefore have high potential for the development of new therapeutics. To be able to screen for potent and selective inhibitors sufficient amounts of protein are required. Here, we present an easy and efficient protocol for the recombinant expression of soluble and active murine cathepsin B and L. For this, we used the strain E. coli SHuffle® T7 Express which is capable of forming disulfide bridges in the cytoplasm. The enzymes were purified by immobilized nickel ion-affinity chromatography. Using different constructs and media, expression levels were significantly improved and expression yields of 80 ± 2 mg L⁻¹ for procathepsin B, which is 16-fold better than previously reported expression yields for procathepsin B, and 37 ± 2 mg L⁻¹ for procathepsin L, were achieved. After activation with dithiothreitol at slightly acidic pH, in vitro kinetic parameters of both cathepsins were determined using the commonly used synthetic substrates Arg-Arg-AMC or Phe-Arg-AMC. Moreover, to investigate the impact of the short C-terminal propeptide of procathepsin B, it was deleted by site-directed mutagenesis, the shortened target protein was expressed and purified, activated in vitro, and its activity was similar to the variant bearing this C-terminal propeptide.

• Recombinant gene expression of cathepsin B and L in E. coli SHuffle® T7 Express

• Soluble cathepsin expression with high expression yields

• Investigation of the short C-terminal propeptide of cathepsin B

Tetracycline (Tc) antibiotics, a class of synthetically produced broad-spectrum antimicrobial drugs, have been widely used in animal husbandry, leading to their widespread presence in animal-derived foods. However, misuse, overuse, and non-compliance with withdrawal periods in animal farming have resulted in excessive Tc residues in these foods, which can cause various adverse reactions in humans, induce bacterial resistance, and pose a significant threat to public health. Consequently, the detection of Tc antibiotic residues in animal-derived food has become a critical issue. This study aims to establish a novel method for quantifying Tc residues in animal-derived food using quantum dots (QDs) fluorescence immunoassay (FLISA). The developed method was optimized to achieve a detection limit of 0.69 ng/mL and a quantitative detection range of 1.30 ~ 59.22 ng/mL. The applicability of the method was demonstrated by successfully determining Tc residues in pork, chicken, fish, milk, eggs, and honey samples spiked with Tc standard solutions, yielding recoveries ranging from 94.01% to 110.19% and relative standard deviations between 1.10% and 11.39%. The significance of this study lies in its potential to provide a rapid and reliable approach for monitoring Tc residues in animal-derived food products, thereby contributing to the enhancement of food safety monitoring practices.

• Screen out tetracycline-specific blocking monoclonal antibodies

• The quantitative detection has high specificity and sensitivity

• This method can be a useful tool for laboratories or testing facilities

Metschnikowia, the single-cell yeast form, is a genus of 85 species in the Saccharomycetales order that developed in both aquatic and terrestrial ecosystems after being found in 1899. This yeast is commonly used to control microbial populations in many biological and artificial conditions, such as fermentation. However, current study of Metschnikowia is limited to biological control features rather than researching on lucrative sectors such as beverage production, bioconversion manufacturing, cosmetics, and the pharmaceutical industry. This review summarizes numerous possible applications of Metschnikowia in human life, including potential secondary metabolites in industrial fields such as cosmetics and pharmaceuticals. Furthermore, Metschnikowia-yeast interaction is mentioned as a potential area for further exploration in terms of co-cultured microbes as biocontrol. Since Metschnikowia yeast arose in a variety of ecosystems, more discussion will be held regarding the interactions between Metschnikowia and their surroundings, particularly in fruits. Finally, the current regulatory challenges of Metschnikowia-based products are examined, and future research opportunities on Metschnikowia utilization are presented.

• Utilization of Metschnikowia genus in various human aspects.

• Promising secondary metabolites produced by Metschnikowia.

• Challenge and opportunity on developing Metschnikowia-based products.

White-rot fungi, such as Phanerochaete chrysosporium, play a crucial role in biodegrading lignocellulosic biomass including cellulose, hemicellulose, and lignin. These fungi utilise various extracellular and intracellular enzymes, such as lignin peroxidases, manganese peroxidases, versatile peroxidases, monooxygenases, and dioxygenases, to degrade lignin and lignin-derived aromatics, thereby significantly contributing to the global carbon cycle with potential applications in industrial bioprocessing and bioremediation. Although the metabolism of lignin fragments in P. chrysosporium has been studied extensively, the enzymes involved in fragment conversion remain largely unknown. This review provides an overview of the current knowledge regarding the metabolic pathways of lignin and its fragments by white-rot fungi. Recent studies have elucidated the intricate metabolic pathways and regulatory mechanisms of lignin-derived aromatic degradation by focusing on flavoprotein monooxygenases, intradiol dioxygenases, homogentisate dioxygenase-like proteins, and cytochrome P450 monooxygenases. Metabolic regulation of these enzymes demonstrates the adaptability of white-rot fungi in degrading lignin and lignin-derived aromatics. The interplay between the central metabolic pathways, haem biosynthesis, and haem-dependent NAD(P)H regeneration highlights the complexity of lignin degradation in white-rot fungi. These insights improve our understanding of fungal metabolism and pave the way for future studies aimed at leveraging these fungi for sustainable biotechnological applications.

• White-rot fungi use enzymes to degrade lignin, and play a role in the carbon cycle.

• Oxygenases are key enzymes for converting lignin-derived aromatics.

• White-rot fungi adapt to metabolic changes by controlling the TCA/glyoxylate bicycle.

Rampant caries is identified by rapid onset, severe decay affecting multiple surfaces, and early pulp infection. This case–control study was conducted to investigate the disparities in oral microbiota between children affected by rampant caries and their caries-free counterparts. A total of 88 preschool children, with matched distribution of sex and age in both the case and control groups, participated in this study. Children’s oral health–related behaviors were reported by parents, salivary pH levels were assessed using a portable pen-type pH meter, and supragingival dental plaque was analyzed by 16S rRNA gene sequencing. Children with rampant caries exhibited lower salivary pH levels, poorer toothbrushing habits, and more frequent consumption of sugary snacks. Veillonella, enriched in caries-free children, showed a positive correlation with salivary pH levels and a negative correlation with candy consumption. Conversely, Fusobacterium and Neisseria, more abundant in children with rampant caries, positively correlated with the frequency of candy consumption. Furthermore, Streptococcus mutans, Porphyromonas gingivalis, and Bacteroides acidifaciens were identified as potential oral microbiome markers for differentiating preschoolers with rampant caries from their caries-free peers. B. acidifaciens, typically found in the gut, has been rarely reported in the field of oral health. More well-designed cohort studies are recommended to elucidate the mechanisms through which gut microbiota influences rampant caries in pediatric patients and offer insights into effective strategies for caries management in young children.

• Lower salivary pH levels in children with rampant caries.

• Biomarkers for predicting rampant caries.

• Impact of oral health–related behaviors on oral microbiota.

Coelimycin (CPK) producer Streptomyces coelicolor A3(2) is a well-established model for the genetic studies of bacteria from the genus Streptomyces, renowned for their ability to produce a plethora of antibiotics and other secondary metabolites. Expression regulation of natural product biosynthetic gene clusters (BGCs) is highly complex, involving not only regulatory proteins, like transcription factors, but also the products of the biosynthetic pathway that may act as ligands for some regulators and modulate their activity. Here, we present the evidence that intracellular CPK precursor(s) (preCPK) is involved in a negative feedback loop repressing the CPK BGC. Moreover, we provide a characterization of the cluster-encoded efflux pump CpkF. We show that CpkF is essential for the extracellular CPK production. In order to track down which CPK compounds — intra- or extracellular — are the ones responsible for the feedback signal, a luciferase-based reporter system was applied to compare the activity of 13 CPK gene promoters in the wild-type (WT) and two mutated strains. The first strain, lacking the CPK-specific exporter CpkF (ΔcpkF), was unable to produce the extracellular CPK. The second one did not produce any CPK at all, due to the disruption of the CpkC polyketide synthase subunit (ΔcpkC). All tested promoters were strongly upregulated in ΔcpkC strain, while in the ΔcpkF strain, promoter activity resembled the one of WT. These results lead to the conclusion that the CPK polyketide acts as a silencer of its own production. Supposedly this function is exerted via binding of the preCPK by an unidentified regulatory protein.

•Intracellular coelimycin precursor takes part in a negative cpk cluster regulation

•CpkF exporter is essential for the extracellular coelimycin production

•Simple method for the analysis of coelimycin P2 production in agar medium

Violacein is a natural indole-derived purple pigment of microbial origin that has attracted attention for its remarkable biological properties. Due to its poor solubility in aqueous media, most studies of this pigment use extracts of the compound obtained with common solvents. Violacein is also transported in bacterial extracellular vesicles (EVs) and transferred via this type of carrier remains stable in an aqueous environment. This paper is the first to present an in-depth study of Janthinobacterium lividum EVs as violacein carriers. J. lividum EVs were studied for their contribution to violacein translocation, size, morphology and protein composition. The production of violacein encapsulated in EVs was more efficient than the intracellular production of this compound. The average size of the violacein-containing EVs was 124.07 ± 3.74 nm. Liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) revealed 932 proteins common to three independent EVs isolations. The high proportion of proteins with intracellular localisation, which are involved in many fundamental cellular processes, suggests that J. lividum EVs could be generated in a cell lysis model, additionally stimulated by violacein production. Using human keratinocytes and melanoma cell lines, it was confirmed that J. lividum EVs are able to react with and deliver their cargo to mammalian cells. The EVs-delivered violacein was shown to retain its activity against melanoma cells, and the dose and timing of treatment can be selected to target only cancer cells. The characterisation of J. lividum EVs, described in the following paper, represents a milestone for their future potential anticancer application.

• This report focuses on the investigation of Janthinobacterium lividum EVs as a new delivery vehicle for violacein, a compound with a previously demonstrated broad spectrum of activity.

• EVs were characterised for size, morphology and protein composition.

• Studies on human keratinocytes and a melanoma cell model confirmed that the activity of violacein applied in the encapsulated form of EVs is similar to that of its organic solvent extract, but their production is much more environmentally friendly.

Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering.

• Blue LED light hinders growth in CHO DP-12 cells

• Antioxidants protect to a certain degree from blue light effects

• Illumination with blue LED light raises cell-specific productivity