ChemBioChem最新文献

A sucrose-utilization pathway was developed in Pseudomonas putida using sacC from Mannheimia succiniciproducens, which encodes a β-fructofuranosidase that hydrolyzes sucrose into glucose and fructose. Excretion of β-fructofuranosidase into the culture medium was confirmed via western blot analysis. In nitrogen-limited cultivation, P. putida expressing SacC produced 10.52 wt % medium-chain-length polyhydroxyalkanoate (MCL-PHA), while P. putida expressing SacC along with poly(3-hydroxybutyrate) [P(3HB)] biosynthesis genes produced 9.16 wt % P(3HB) from sucrose. Batch and fed-batch cultures of recombinant P. putida suggested that the glucose and fructose derived from sucrose can be completely utilized for cell growth and P(3HB) production. In fed-batch cultures, sucrose supplied into the fermentor to maintain its concentration around 20 g/L was rapidly hydrolyzed into glucose and fructose supporting the production of 30.2 g/L P(3HB) with 38.1 wt %. The engineered P. putida reported herein can facilitate the production of PHAs from sucrose, an abundant and inexpensive carbon source.

The malaria parasite Plasmodium falciparum affects the lives of millions of people worldwide every year. The detection of replicating parasites within human red blood cells is of paramount importance, requiring appropriate diagnostic tools. Herein, we design and apply a silicon rhodamine-fused glibenclamide (SiR-glib). We first test this far-red fluorescent, fluorogenic and endoplasmic reticulum-targeting sulfonylurea in mammalian cells and pancreatic islets, before characterizing its labeling performance in red blood cells infected with the asexual developmental stages of Plasmodium falciparum. We further combine SiR-glib with a portable smartphone-based microscope to easily and rapidly identify parasitized red blood cells, providing proof of principle for diagnostic use in rural endemic areas without major healthcare facilities.

RNA switches regulated by specific inducer molecules have become a powerful synthetic biology tool for precise gene regulation in mammalian systems. The engineered RNA switches can be integrated with natural RNA-mediated gene regulatory functions as a modular and customizable approach to probe and control cellular behavior. RNA switches have been used to advance synthetic biology applications, including gene therapy, bio-production, and cellular reprogramming. This review explores recent progress in the design and functional implementation of synthetic riboswitches in mammalian cells based on diverse RNA regulation mechanisms by highlighting recent studies and emerging technologies. We also discuss challenges such as off-target effects, system stability, and ligand delivery in complex biological environments. In conclusion, this review emphasizes the potential of synthetic riboswitches as a platform for customizable gene regulation in diverse biomedical applications.

RNA modifications are involved in numerous biological processes and vary in different cell types. Methylation is the most widespread type of RNA modification and occurs via S-adenosyl-L-methionine (SAM). We recently developed a metabolic labeling approach based on intracellular formation of a clickable SAM analog (SeAdoYn) and demonstrated its use in mapping methyltransferase (MTase) target sites in mRNA from HeLa cells. Here we investigate how metabolic labeling via the clickable SAM analog modifies four different nucleosides in RNA of HEK293T in comparison to HeLa cells. We find that HEK293T cells retain higher cell viability upon feeding the clickable metabolic SAM precursor. In poly(A)⁺ RNA we find high A_prop/A levels (0.04 %) and in total RNA (but not poly(A)⁺ RNA) we detect prop³C, which had not been detected previously in HeLa cells. We discuss the findings in the context of data from the literature with respect to mRNA half-lives in cancer and non-cancer cell lines and suggest that CMTr2 is most likely responsible for the high A_prop level in poly(A)⁺ RNA.

Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme in de novo Ser biosynthesis. Numerous metabolic pathways rely on Ser as a precursor, most notably one-carbon metabolism, glutathione biosynthesis, and de novo nucleotide biosynthesis. To facilitate proliferation, many cancer cells shunt glycolytic flux through this pathway, placing PHGDH as a metabolic liability and feasible therapeutic target for the treatment of cancer. Herein, we demonstrate the post-translational modification (PTM) of PHGDH by lactoylLys. These PTMs are generated through a non-enzymatic acyl transfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Knockout of the primary LGSH regulatory enzyme, glyoxalase 2 (GLO2), results in increased LGSH and resulting lactoylLys modification of PHGDH. These PTMs reduce enzymatic activity, resulting in a marked reduction in intracellular Ser. Using stable isotope tracing, we demonstrate reduced flux through the de novo Ser biosynthetic pathway. Collectively, these data identify PHGDH as a target for modification by lactoylLys, resulting in reduced enzymatic activity and reduced intracellular Ser.

Fibrinogen is a protein involved in the haemostasis process playing a central role by forming the fibrin clot. An understanding of protein structure is vital to determining biological function. Despite many studies on the fibrin polymerization process, its molecular mechanism remains elusive mainly due to the absence of a full-length three-dimensional model of human fibrinogen. Amino- and carboxyl-terminal regions of the three pairs of chains that form this molecule are missing in the crystallographic structure, being the carboxyl-terminal of the Aα chain the most affected with a section of more than 400 amino acids missing. To have a full structure of the fibrinogen molecule would allow the creation of a model of protofibril, shedding light into the fibrin formation process through computational techniques such as molecular dynamics simulations. Absent regions were explored using homology modelling and coarse-grained molecular dynamics simulations. Later on, the model was refined and stabilized with atomistic molecular dynamic simulations. In the present study, we obtained the first realistic full-length structure of fibrinogen, with features in accordance with previous results obtained by experimental techniques.

Recognition of glycans by simple synthetic receptors is a key issue in supramolecular chemistry, endowed with relevant implications in glycobiology and medicine. In this context, glycoproteins featuring N-glycans represent an important biological target, because they are often exploited by enveloped viruses in adhesion and infection processes. However, a direct evidence for their recognition by a synthetic receptor targeting N-glycans is still missing in the literature. Using a combination of glycoengineering and mass spectrometry techniques, we present here the direct evidence of biomimetic recognition of complex-type N-glycans exposed on the receptor-binding domain (RBD) of the wild-type spike protein of SARS-CoV-2 by a biologically active, synthetic receptor.

Ascomycete fungi of the genus Fusarium are found in manifold ecological niches and thus pursue several lifestyles. On average, individual Fusarium species have the genetic capability to produce 50 natural products (NPs), which are in general thought to improve the fungus's fitness in defined environments. This also includes NPs with toxic potential (mycotoxins) contaminating food and feed sources. Recent research has shown that the production of NPs is tightly regulated on the transcriptional level and depends on the delicate balance between the deposition and removal of histone marks. Within this study, we show that the expression of the prior cryptic Fusarium PKS16 biosynthetic gene cluster (BGC) greatly depends on modifications at histone 3 lysine 27 (H3K27). By combining molecular-, chemical-, and bioinformatic analyses we show that the PKS16 BGC from F. fujikuroi B14 (FfB14) consists of nine genes, including a positively acting pathway-specific transcription factor, which although absent in some fusaria, functions in activating other PKS16 cluster genes. Moreover, we linked the PKS16 BGC to the biosynthesis of proliferapyrone (PRO) B, an isomer of the recently isolated PRO A.

In scientific communication about three-dimensional structures, creating two-dimensional representations is standard practice. These representations often suffer from the drawback of losing potential information due to dimensionality reduction. Several options exist to present, share and publish 3D figures, however based on recent publications they are not widely utilized. Here we present simple ways to preserve the three-dimensionality of the structure by the creation of a custom-made model in GLTF format that is generated in the same workflow as the conventional figures. They can be published alongside a given manuscript with minimal additional effort to the authors, but a huge impact on the communicative power of the manuscript concerning the three-dimensional features of the reported structures. The scripts we adapted and published for this purpose open up new possibilities for the illustrator and allow the viewer to access the full three-dimensionality of the published structure. In future, this can simplify the publication process of protein structures or other models and be a valuable tool for scientific communication in digital or printed form.

Cytoplasmic glutathione S-transferase (GST) is a key enzyme in cellular detoxification, catalysing the nucleophilic attack of glutathione (GSH) with toxic electrophilic substrates to produce less harmful compounds, thus aiding cellular detoxification. Studies have shown that GST is closely associated with the development of resistance to chemotherapeutic drugs, pesticides, herbicides and antibiotics, and the development of drug resistance in organisms poses new challenges in areas such as environmental protection and tumour therapy. In order to clarify the mechanism of GST in the development of drug resistance and detect the content of GST more accurately, this paper summarized the mechanism of GST on the development of drug resistance in different organisms, the types and research progress of organic small molecule fluorescence probes for GST imaging detection are introduced.