Search Results (428)

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution. Full article

Background: Hydrogels are 3D networks of hydrophilic polymers with various biomedical applications, including tissue regeneration, wound healing, and localized drug delivery. Hydrogel conjugation links therapeutic agents to a hydrogel network, creating a delivery system with adjustable and flexible hydrogel properties and drug activity, allowing for controlled release and enhanced drug stability. Conjugating therapeutic agents to hydrogels provides innovative delivery formats, including injectable and sprayable dosage forms, which facilitate localized and long-lasting delivery. This approach enables non-viral therapeutic methods, such as insertional mutagenesis, and minimally invasive drug administration. Scope and Objectives: While numerous reviews have analyzed advancements in hydrogel synthesis, characterization, properties, and hydrogels as a drug delivery vehicle, this review focuses on hydrogel conjugation, which enables the precise functionalization of hydrogels with small molecules and macromolecules. Subsequently, a description and discussion of several bio-conjugated hydrogel systems, as well as binding motifs (e.g., “click” chemistry, functional group coupling, enzymatic ligation, etc.) and their potential for clinical translation, are provided. In addition, the integration of therapeutic agents with nucleic acid-based hydrogels can be leveraged for sequence-specific binding, representing a leap forward in biomaterials. Key findings: Special attention was given to the latest conjugation approaches and binding motifs that are useful for designing hydrogel-based drug delivery systems. The review systematically categorizes hydrogel conjugates for drug delivery, focusing on conjugating hydrogels with major classes of therapeutic agents, including small-molecule drugs, nucleic acids, proteins, etc., each with distinct conjugation challenges. The design principles were discussed along with their properties and drug release profiles. Finally, future opportunities and current limitations of conjugated hydrogel systems are addressed. Full article

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae. Full article

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function. Full article

Yeast genetic improvement is entering a transformative phase, driven by the integration of artificial intelligence (AI), big data analytics, and synthetic microbial communities with conventional methods such as sexual breeding and random mutagenesis. These advancements have substantially expanded the potential for innovative re-engineering of yeast, ranging from single-strain cultures to complex polymicrobial consortia. This review compares traditional genetic manipulation techniques with cutting-edge approaches, highlighting recent breakthroughs in their application to beer and wine fermentation. Among the innovative strategies, adaptive laboratory evolution (ALE) stands out as a non-GMO method capable of rewiring complex fitness-related phenotypes through iterative selection. In contrast, GMO-based synthetic biology approaches, including the most recent developments in CRISPR/Cas9 technologies, enable efficient and scalable genome editing, including multiplexed modifications. These innovations are expected to accelerate product development, reduce costs, and enhance the environmental sustainability of brewing and winemaking. However, despite their technological potential, GMO-based strategies continue to face significant regulatory and market challenges, which limit their widespread adoption in the fermentation industry. Full article

The nopal cactus, a plant from the Cactaceae family, holds significant economic and nutritional value for Mexico. This study aimed to enhance the genetic diversity and morphological traits of Opuntia velutina, a species cultivated as a vegetable nopal. A total of 1050 in vitro O. velutina explants were exposed to 15 different doses of gamma radiation from ⁶⁰Co gamma, ranging from 5 to 125 Gy. The lethal dose was above 50 Gy, with an LD₅₀ of 22.8 Gy for stimulating in vitro shoot growth. Shoots derived from doses between 5 and 50 Gy were subjected to in vitro shoot proliferation across four consecutive generations to stabilize morphological traits. Cluster analysis categorized the 178 irradiated shoots into 13 distinct morphological groups (CG1–CG13). Twenty-seven shoots exhibiting significant morphological improvements, such as a 50–100% increase in cladode length, up to a six-fold increase in shoot number, and up to a seven-fold increase in root number, were selected for molecular analysis of genetic diversity. Six primers were used with the Inter Simple Sequence Repeat (ISSR) molecular markers to examine genetic uniformity, yielding 54.5% polymorphic bands, indicating a high level of genetic variation. Both a UPGMA dendrogram and STRUCTURE-based Bayesian analysis confirmed the genetic divergence among the selected mutant lines. Overall, gamma irradiation effectively enhanced both phenotypic and genotypic diversity in O. velutina. This study corroborates that in vitro mutagenesis through gamma radiation is a viable strategy for generating novel genotypes with breeding potential within the Opuntia genus. Full article

Leaf morphology influences photosynthesis, transpiration, and, ultimately, crop yield. To elucidate the molecular regulatory mechanisms underlying narrow leaves in Sorghum bicolor, we identified key DEGs (differentially expressed genes) influencing leaf morphology. The nal6 (the narrow-leaf mutant6) was obtained through 0.1% EMS (ethyl methane sulfonate) chemical mutagenesis of the WT (BTX623). Compared with the WT leaves, there were significant differences in leaf width and length at the flowering stage. A total of 1520 DEGs between the nal6 and WT were screened at the flowering stage based on the transcriptome analysis of sword leaves. KEGG and GO enrichment analyses revealed that DEGs were significantly enriched in pathways such as plant signal transduction, cytokinin biosynthesis, photosynthetic antenna proteins, and secondary metabolite biosynthesis. Further analysis indicated that four DEGs are involved in regulating auxin signaling transduction, thirteen DEGs are involved in regulating zeatin signal transduction, and two DEGs are involved in regulating zeatin biosynthesis. These genes are differentially expressed in nal6, directly affecting the signaling of auxin and zeatin and the biosynthesis of zeatin. Our findings provide a theoretical foundation for understanding the molecular regulation of narrow leaves and breeding ideal plant types in Sorghum bicolor. Full article

The aim of this study was to investigate the biological relevance of predicted sites involved in protein–protein interaction formation by bHLH transcription factors associated with gynoecium development in Arabidopsis (Arabidopsis thaliana). We used AlphaFold2 to generate three-dimensional protein structures of the bHLH proteins SPATULA (SPT), HECATE1 (HEC1), and INDEHISCENT (IND). These structures were subjected to molecular docking using the HawkDock server, enabling the identification of potential interaction sites. PCR-based site-directed mutagenesis was used to modify the predicted interaction sites, followed by testing for protein–protein interaction formation using Bimolecular Fluorescence Complementation (BiFC) assays. Furthermore, these modified versions were overexpressed in Arabidopsis to observe whether gynoecium and fruit development would be affected. BiFC assays with the modified versions revealed a complete loss of the SPT-HEC1 interaction and a strong reduction in the SPT-IND interaction. The overexpression experiments in Arabidopsis showed that the 35S::SPT-4A line exhibited strong phenotypes in the development of the medial tissues of the gynoecium, resulting in reduced seed number and shorter fruits. In the 35S::HEC1-2A line, a reduced seed number and shorter fruits were also observed, but no other obvious defects were observed. Finally, the 35S::IND-3A line was less affected than the 35S::IND line. In the latter, medial tissue development was strongly affected, while in the 35S::IND-3A line, it was only slightly affected; however, a reduced seed number and shorter fruits were observed. In summary, the predicted interaction sites are relevant and, when modified, affect gynoecium development in Arabidopsis. The findings demonstrate that predictive computational tools represent a viable strategy for a deeper understanding of protein–protein interactions. Full article

Rice (Oryza sativa L.) is a stable food for over half of the world population, contributing 50–80% of the daily calorie intake. The completion of rice genome sequencing marks a significant milestone in understanding functional genomics, yet the systematic identification of gene functions remains a bottleneck for rice improvement. Large-scale mutant libraries in which the functions of genes are lost or gained (e.g., through chemical/physical treatments, T-DNA, transposons, RNAi, CRISPR/Cas9) have proven to be powerful tools for the systematic linking of genotypes to phenotypes. So far, using different mutagenesis approaches, a million mutant lines have been established and about 5–10% of the predicted rice gene functions have been identified due to the high demands of labor and low-throughput utilization. DNA-barcoding-based large-scale mutagenesis offers unprecedented precision and scalability in functional genomics. This review summarizes large-scale loss-of-function and gain-of-function mutant library development approaches and emphasizes the integration of DNA barcoding for pooled analysis. Unique DNA barcodes can be tagged to transposons/retrotransposons, DNA constructs, miRNA/siRNA, gRNA, and cDNA, allowing for pooling analysis and the assignment of functions to genes that cause phenotype alterations. In addition, the integration of high-throughput phenotyping and OMICS technologies can accelerate the identification of gene functions. Full article

The demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L-Trp production in Escherichia coli. Firstly, an L-Trp-producing strain was selected and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis to generate a mutant library. This was followed by high-throughput screening using an L-Trp-specific riboswitch and a yellow fluorescent protein (YFP)-based biosensor in a flow cytometric cell sorting (FACS) system. Among the screened mutants, GT3938 exhibited a 1.94-fold increase in L-Trp production. Subsequently, rational metabolic engineering was applied to GT3938 by knocking out the L-Trp intracellular transporter gene (tnaB), enhancing the expression of the aromatic amino acid exporter (YddG) and optimizing precursor supply pathways. The resulting strain, zh08, achieved an L-Trp titer of 3.05 g/L in shake-flask fermentation, representing a 7.71-fold improvement over the original strain. This study demonstrated an effective strategy for industrial strain development by integrating biosensor-assisted, high-throughput screening with rational metabolic engineering. Full article

Comparative studies using lysophosphatidic acid (LPA) and the synthetic agonist, oleoyl-methoxy glycerophosphothionate (OMPT), in cells expressing the LPA₃ receptor revealed differences in the action of these agents. The possibility that more than one recognition cavity might exist for these ligands in the LPA₃ receptor was considered. We performed agonist docking studies exploring the whole protein to obtain tridimensional details of the ligand–receptor interaction. Functional in cellulo experiments using mutants were also executed. Our work includes blind docking using the unrefined and refined proteins subjected to hot spot predictions. Distinct ligand protonation (charge −1 and −2) states were evaluated. One LPA recognition cavity is located near the lower surface of the receptor close to the cytoplasm (Lower Cavity). OMPT displayed an affinity for an additional identification cavity detected in the transmembrane and extracellular regions (Upper Cavity). Docking targeted to Trp102 favored binding of both ligands in the transmembrane domain near the extracellular areas (Upper Cavity), but the associating amino acids were not identical due to close sub-cavities. A receptor model was generated using AlphaFold3, which properly identified the transmembrane regions of the sequence and co-modeled the lipid environment accordingly. These two models independently generated (with and without the membrane) and adopted essentially the same conformation, validating the data obtained. A DeepSite analysis of the model predicted two main binding pockets, providing additional confidence in the predicted ligand-binding regions and support for the relevance of the docking-based interaction models. In addition, mutagenesis was performed of the amino acids of the two detected cavities. In the in cellulo studies, LPA action was much less affected by the distinct mutations than that of OMPT (which was almost abolished). Therefore, docking and functional data indicate the presence of distinct agonist binding cavities in the LPA₃ receptor. Full article

During Bacillus subtilis spore germination/outgrowth, the rehydration of the spore core and activation of aerobic metabolism can generate reactive oxygen species (ROS)-promoted DNA lesions that are repaired via the base excision repair pathway (BER). Accordingly, spores deficient in the AP-endonucleases (APEs) Nfo and ExoA exhibit a delayed outgrowth that is suppressed following disruption of the checkpoint protein DisA. Here, we report that DisA-independent DNA damage checkpoints operate during B. subtilis spore outgrowth. Consistent with this notion, spores lacking Nfo, ExoA, and Nth, which functions as an APE, did not suppress delayed outgrowth following disA disruption. Furthermore, in reference to the ∆nfo ∆exoA ∆nth spores, spores deficient for these APEs and DisA displayed a significantly higher number of oxidative genetic lesions and failed to properly segregate its chromosome during the first round of replication in the outgrowth stage. Finally, we found that DisA promotes low-fidelity repair and replication events, as revealed by DNA-alkaline gel electrophoresis (AGE) as well as spontaneous and H₂O₂-promoted Rif^R mutagenesis. Overall, our results unveil the existence of DisA-independent DNA damage checkpoint(s) that are activated by genomic lesions of an oxidative nature during spore germination and outgrowth, ensuring a proper transition to vegetative growth. Full article

In this study, we established a mutagenesis and high-throughput screening system to select a high-yielding glutathione (GSH)-producing strain of Saccharomyces cerevisiae. The parent strain was mutated by atmospheric and room temperature plasma (ARTP) technology and cultivated using ethionine plate cultivation. Subsequently, high-throughput screening was performed using liquid deep microtiter plates (MTPs) for cultivation and a microplate reader for rapid GSH detection. The results demonstrated the successful selection of a stable mutant strain, S-272, which exhibited significantly enhanced GSH production. Fermentation validation in 5 L bioreactors revealed that S-272 achieved a 14.7% higher final GSH concentration and a 19.5% higher intracellular GSH content compared to the parent strain. The improved performance of S-272 was attributed to enhanced ethanol utilization, elevated activity of γ-glutamylcysteine synthetase (γ-GCS), and increased intracellular trehalose content. This study presents an effective strategy for developing high GSH-yield strains using ARTP complex mutagenesis technology combined with high-throughput screening. Full article

Background/Objectives: The AXL receptor tyrosine kinase is a promising therapeutic target in solid tumors, yet conventional viral vector-engineered CAR-T cells face critical limitations, including risks of insertional mutagenesis and immunogenicity from murine-derived single-chain variable fragments (scFvs). This study aimed to develop and evaluate mRNA-engineered fully human AXL CAR-T (^mfhAXL CAR-T) cells as a safer, scalable alternative for solid tumor immunotherapy. Methods:^mfhAXL CAR-T cells were generated via electroporation-mediated delivery of in vitro transcribed mRNA encoding a fully human AXL-specific CAR. CAR expression kinetics and T-cell viability were quantified by flow cytometry. Antitumor activity was assessed through in vitro co-cultures with AXL-positive lung and pancreatic cancer cells, measuring cytotoxicity, cytokine secretion, and specificity. In vivo efficacy was evaluated in a lung cancer xenograft mouse model, with tumor volume and body weight monitored over 14 days. Results: Flow cytometry confirmed transient but high CAR expression (>90% at 24 h) with preserved T-cell viability (>90%). In vitro, ^mfhAXL CAR-T cells exhibited dose-dependent cytotoxicity and antigen-specific cytokine secretion. In vivo, four administrations of ^mfhAXL CAR-T cells suppressed tumor growth without body weight loss. Conclusions: The mRNA-electroporated ^mfhAXL CAR-T platform enables cost-effective, large-scale production, offering a safer alternative to viral vector-based approaches by eliminating risks of insertional mutagenesis and immunogenicity. Full article