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13 pages, 1186 KiB  
Article
Targeting the Cell Wall Salvage Pathway: Dual-Enzyme Inhibition of AmgK and MurU as a Strategy Against Antibiotic Resistance
by Hwa Young Kim, Seri Jo, Mi-Sun Kim and Dong Hae Shin
Int. J. Mol. Sci. 2025, 26(15), 7368; https://doi.org/10.3390/ijms26157368 - 30 Jul 2025
Viewed by 218
Abstract
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 ( [...] Read more.
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
(This article belongs to the Section Molecular Microbiology)
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34 pages, 4483 KiB  
Review
A Beautiful Bind: Phage Display and the Search for Cell-Selective Peptides
by Babak Bakhshinejad and Saeedeh Ghiasvand
Viruses 2025, 17(7), 975; https://doi.org/10.3390/v17070975 - 12 Jul 2025
Viewed by 569
Abstract
Phage display has advanced the discovery of peptides that selectively bind to a wide variety of cell surface molecules, offering new modalities to modulate disease-related protein–protein interactions (PPIs). These cell-binding peptides occupy a unique pharmaceutical space between small molecules and large biologics, and [...] Read more.
Phage display has advanced the discovery of peptides that selectively bind to a wide variety of cell surface molecules, offering new modalities to modulate disease-related protein–protein interactions (PPIs). These cell-binding peptides occupy a unique pharmaceutical space between small molecules and large biologics, and their growing popularity has opened up new avenues for targeting cell surface proteins that were previously considered undruggable. This work provides an overview of methods for identifying cell-selective peptides using phage display combinatorial libraries, covering in vitro, ex vivo, and in vivo biopanning approaches. It addresses key considerations in library design, including the peptide conformation (linear vs. cyclic) and length, and highlights examples of clinically approved peptides developed through phage display. It also discusses the on-phage chemical cyclization of peptides to overcome the limitations of genetically encoded disulfide bridges and emphasizes advances in combining next-generation sequencing (NGS) with phage display to improve peptide selection and analysis workflows. Furthermore, due to the often suboptimal binding affinity of peptides identified in phage display selections, this article discusses affinity maturation techniques, including random mutagenesis and rational design through structure–activity relationship (SAR) studies to optimize initial peptide candidates. By integrating these developments, this review outlines practical strategies and future directions for harnessing phage display in targeting challenging cell surface proteins. Full article
(This article belongs to the Special Issue The Application of Viruses to Biotechnology 3.0)
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17 pages, 7108 KiB  
Article
Transcriptome Differential Expression Regulation Analysis of the Narrow-Leaf Mutant of Sorghum Bicolor
by Jinhong Li, Yiwei Wang, Yuche Zhao, Shirui Zhang, Chunyu Wang, Ling Cong, Yanpeng Zhang, Shuang Gang and Xiaochun Lu
Agronomy 2025, 15(6), 1432; https://doi.org/10.3390/agronomy15061432 - 12 Jun 2025
Viewed by 845
Abstract
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 [...] Read more.
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
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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17 pages, 1924 KiB  
Article
Development of a High-Performance Trichoderma Mutant for Enhanced Cellulase Production Through UV-Induced Random Mutagenesis
by Seungjun Kim, Iksu Ha, Yun-Yeong Lee, Junseo Lee and Jeonghee Yun
J. Fungi 2025, 11(6), 439; https://doi.org/10.3390/jof11060439 - 9 Jun 2025
Viewed by 902
Abstract
Ultraviolet (UV)-induced mutagenesis is a cost-effective and straightforward technique for introducing random genetic variations without the use of chemical reagents or genetic engineering. It is commonly employed to enhance enzyme activity in industrial trains. In this study, Trichoderma sp. was exposed to UV [...] Read more.
Ultraviolet (UV)-induced mutagenesis is a cost-effective and straightforward technique for introducing random genetic variations without the use of chemical reagents or genetic engineering. It is commonly employed to enhance enzyme activity in industrial trains. In this study, Trichoderma sp. was exposed to UV radiation at varying distances (4, 9, and 13 cm) and durations (2, 4, 6, and 8 min) to induce mutations. The activities of endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH) were assessed following treatment. The 4 cm exposure distance yielded the highest enhancement, with EG, BGL, and CBH activities increasing 1.5-, 1.3-, and 0.9-fold, respectively. When the distance was fixed at 4 cm, the optimal exposure time was identified as 4 min, yielding further enhancements of 1.9-, 1.6-, and 1.4-fold, respectively. The resulting mutant, designated Mut-4, was scaled up in a 10-L bioreactor to assess its industrial applicability. Mut-4 retained its enhanced performance, achieving 1.9-, 2.0-, and 1.4-fold enhancements in EG, BGL, and CBH activities, respectively, compared with the original strain. These findings indicate that combining UV-induced mutagenesis with basic screening is an effective strategy for enhancing cellulolytic enzyme production, representing a promising approach for lignocellulosic biomass conversion. Full article
(This article belongs to the Section Fungi in Agriculture and Biotechnology)
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14 pages, 3449 KiB  
Article
Characterization of Terpene Synthases Reveals the Diversity of Terpenoids in Andrographis paniculata
by Junhao Tang, Ying Ma, Yujun Zhao, Xiaohui Ma and Jian Wang
Molecules 2025, 30(10), 2208; https://doi.org/10.3390/molecules30102208 - 18 May 2025
Viewed by 657
Abstract
Terpenoids have significant biological activity and good clinical efficacy and are important for defence and physiological regulation in plants. Andrographolide and similar labdane-related diterpenoids have been isolated and characterized as the main medicinal constituents of drugs from Andrographis paniculata. To better study [...] Read more.
Terpenoids have significant biological activity and good clinical efficacy and are important for defence and physiological regulation in plants. Andrographolide and similar labdane-related diterpenoids have been isolated and characterized as the main medicinal constituents of drugs from Andrographis paniculata. To better study the diversity of terpenoids of A. paniculata, a total of 39 ApTPSs were screened, and 27 full-length genes encoding ApTPSs were obtained. The results showed that ApTPS4 could convert GGPP to ent-CPP and that ApTPS5 could convert ent-CPP to kaurene. This study first identified six sesquiterpene synthases with biological activity and also indicated the presence of sesquiterpenes with multiple skeletons in A. paniculata. The increase in the number of ent-copalyl diphosphate synthases and the loss of biological function by most sesquiterpene synthases and monoterpene synthases may explain why diterpenoids are the main specific metabolites in A. paniculata compared with the metabolites produced by AtTPSs found in the Arabidopsis thaliana genome. As revealed by site-directed mutagenesis, 533Val of ApTPS16 is an important site for maintaining the single main product capability, and 534Tyr of ApTPS17 may also be more important. The ApTPS17 Y534V mutation caused it to lose its main biological function. This study characterized a novel ent-copalyl diphosphate synthase and six sesquiterpene synthases. This provided evidence for the existence of other terpenoids and revealed the diversity of chemical components, providing a reference for future pharmacological research for A. paniculata. Full article
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21 pages, 2401 KiB  
Review
Large-Scale Rice Mutant Establishment and High-Throughput Mutant Manipulation Help Advance Rice Functional Genomics
by Eyob Kassaye Wolella, Zhen Cheng, Mengyuan Li, Dandan Xia, Jianwei Zhang, Liu Duan, Li Liu, Zhiyong Li and Jian Zhang
Plants 2025, 14(10), 1492; https://doi.org/10.3390/plants14101492 - 16 May 2025
Viewed by 1468
Abstract
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 [...] Read more.
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
(This article belongs to the Special Issue Crop Improvement by Modern Breeding Strategies)
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20 pages, 8015 KiB  
Article
Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production
by Sibtain Ahmed, Hina Andaleeb, Aqsa Aslam, Junaid Ahmad Raza, Sheikh Muhammad Yahya Waseem, Atayyaba Javaid and Chand Talib
Fermentation 2025, 11(5), 274; https://doi.org/10.3390/fermentation11050274 - 11 May 2025
Viewed by 958
Abstract
Fungal species secrete various enzymes and are considered the primary sources of industrially important cellulases. Cellulases are essential natural factors for cellulose degradation and have attracted significant interest for multiple applications. However, reducing the cost and enhancing cellulase production remains a significant challenge. [...] Read more.
Fungal species secrete various enzymes and are considered the primary sources of industrially important cellulases. Cellulases are essential natural factors for cellulose degradation and have attracted significant interest for multiple applications. However, reducing the cost and enhancing cellulase production remains a significant challenge. Mutagenesis has opened a new window for enhancing enzyme secretion by modifying the organism’s genome. In this study, cellulases from Alternaria citri were produced and characterized, and the optimization for ideal fermentation conditions was performed for three types of cellulases (endoglucanase, exoglucanase, and β-glucosidase) by a wild-type (A. citri) and a mutant strain (A. citri 305). Ethyl methanesulfonate, a chemical mutagen, was used to enhance cellulase production by A. citri. The results demonstrate the improved cellulolytic ability of the mutant strain A. citri 305 utilizing lignocellulosic waste substances, particularly, orange-peel powder, wheat straw, sugarcane bagasse, and sawdust, making this study economically valuable. This evokes the potential for multi-dimensional applications in enzyme production, waste degradation, and biofuel generation. This study highlights that the activity of cellulases to hydrolyze various lignocellulosic substrates is enhanced after mutagenesis. Full article
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33 pages, 6152 KiB  
Review
A Comprehensive Guide to Enzyme Immobilization: All You Need to Know
by Marina Simona Robescu and Teodora Bavaro
Molecules 2025, 30(4), 939; https://doi.org/10.3390/molecules30040939 - 18 Feb 2025
Cited by 17 | Viewed by 8226
Abstract
Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including [...] Read more.
Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including adsorption, entrapment, encapsulation, and covalent bonding, as well as advanced site-specific methods that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These techniques enable precise control over enzyme orientation and interaction with carriers, optimizing catalytic activity and reusability. Key findings highlight the impact of immobilization on improving enzyme performance under various operational conditions and its role in reducing process costs through enhanced stability and recyclability. The review presents numerous practical applications of immobilized enzymes, including their use in the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and in environmental biotechnology for wastewater treatment and dye degradation. Despite the significant advantages, challenges such as activity loss due to conformational changes and mass transfer limitations remain, necessitating tailored immobilization protocols for specific applications. The integration of immobilization with modern biotechnological advancements, such as site-directed mutagenesis and recombinant DNA technology, offers a promising pathway for developing robust, efficient, and sustainable biocatalytic systems. This comprehensive guide aims to support researchers and industries in selecting and optimizing immobilization techniques for diverse applications in pharmaceuticals, food processing, and fine chemicals. Full article
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18 pages, 3468 KiB  
Review
Environmental Fate, Ecotoxicity, and Remediation of Heterocyclic Pharmaceuticals as Emerging Contaminants: A Review of Long-Term Risks and Impacts
by Oussama Baaloudj, Laura Scrano, Sabino Aurelio Bufo, Lee-Ann Sade Modley, Filomena Lelario, Angelica Rebecca Zizzamia, Lucia Emanuele and Monica Brienza
Organics 2025, 6(1), 1; https://doi.org/10.3390/org6010001 - 2 Jan 2025
Cited by 3 | Viewed by 2509
Abstract
Heterocyclic pharmaceuticals are emerging contaminants due to their toxic, carcinogenic nature and detrimental impact on the natural ecosystem. These compounds pose a significant environmental concern given their widespread use in medical therapy, constituting over 90% of new medications. Their unique chemical structure contributes [...] Read more.
Heterocyclic pharmaceuticals are emerging contaminants due to their toxic, carcinogenic nature and detrimental impact on the natural ecosystem. These compounds pose a significant environmental concern given their widespread use in medical therapy, constituting over 90% of new medications. Their unique chemical structure contributes to their persistence in various environmental matrices, necessitating urgent measures to mitigate their risks. This review comprehensively examines the sources, environmental fate, toxicity, and long-term risks associated with heterocyclic pharmaceuticals, proposing potential remediation strategies. The article commences with an overview of the diverse types of heterocyclic pharmaceuticals and their applications, focusing on compounds containing heteroatoms such as nitrogen, oxygen, and sulfur. Subsequently, it explores the sources and pathways through which these pollutants enter the environment, including wastewater discharge, agricultural runoff, improper disposal, resistance to biodegradation, and bioaccumulation. The toxic effects and long-term consequences of exposure to heterocyclic pharmaceuticals are then discussed, encompassing neurotoxicity, genotoxicity, mutagenesis, cardiovascular and metabolic toxicity, carcinogenicity, and teratogenesis. Additionally, this review summarizes various remediation strategies and treatment solutions aimed at reducing the environmental impact of these compounds, drawing insights from the literature. The research concludes by identifying critical areas for future research, emphasizing the urgent need for more effective remediation strategies to address the growing concern posed by these emerging contaminants. Full article
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16 pages, 10944 KiB  
Article
Targeted Directed Evolution of an α-L-Rhamnosidase on Hesperidin Through Six-Codon Combinatorial Mutagenesis
by Bingbing Wu, Xueting Hou, Na Han, Xinfeng Li, Bin-Chun Li and Guo-Bin Ding
Catalysts 2024, 14(12), 935; https://doi.org/10.3390/catal14120935 - 18 Dec 2024
Viewed by 899
Abstract
Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize [...] Read more.
Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize the labor-determining screening effort remains a challenge. Herein, we propose the six-codon combinatorial mutagenesis (SCCM) strategy by using the BMT degeneracy codons encoding six amino acids with different chemical properties as the building blocks for the randomization of the amnio acid motif. SCCM requires only a small library of 646 clones for 95% coverage at the three-residue motif compared to conventional NNK degeneracy codons encoding all 20 canonical amino acids and requiring the screening of nearly 100,000 clones. SCCM generates a suitable number of mutant libraries, providing a new strategy for reducing the screening workload of saturated combination mutations in enzyme engineering. Using this approach, the α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron had been successfully engineered for improving the hydrolytic activity on natural flavonoid diglycoside hesperidin via targeted directed evolution at the motifs positioning the entrance of the substrate-binding pocket. The results indicate that the conversion rates of the four mutants on hesperidin were increased by more than 30% compared with the wild type using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of the mutant TM1-6-F5 was 1.4-fold higher than that of the wild type. Full article
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16 pages, 3208 KiB  
Article
OCTN1 (SLC22A4) as a Target of Heavy Metals: Its Possible Role in Microplastic Threats
by Luana S. Brunetti, Mariafrancesca Scalise, Raffaella Scanga, Lara Console, Michele Galluccio, Mauro F. La Russa, Lorena Pochini and Cesare Indiveri
Int. J. Mol. Sci. 2024, 25(23), 13218; https://doi.org/10.3390/ijms252313218 - 9 Dec 2024
Cited by 1 | Viewed by 1379
Abstract
Microplastics represent a threat due to their ability to enter the food chain, with harmful consequences for living organisms. The riskiness of these particles is also linked to the release of other contaminants, such as heavy metals. Solute Carriers (SLCs) represent eminent examples [...] Read more.
Microplastics represent a threat due to their ability to enter the food chain, with harmful consequences for living organisms. The riskiness of these particles is also linked to the release of other contaminants, such as heavy metals. Solute Carriers (SLCs) represent eminent examples of first-level targets of heavy metals due to their localization on the cell surface. Putative targets of heavy metals are the organic cation transporters that form a sub-clade of the SLC22 family. Besides the physiological role in the absorption/release of endogenous organic cations, these transporters are crucial in drug disposition and their interaction with xenobiotics. In this work, the human SLC22A4, commonly known as OCTN1, was used as a benchmark to test interactions with heavy metals released by microplastics, exploiting the proteoliposome tool. The potency of metals to interfere with the OCTN1 function has been evaluated by measuring IC50 values calculated in the micromolar range. The molecular mechanism of interaction has been defined using site-directed mutagenesis and computational analyses. Finally, some chemical and physiological thiol-reacting compounds show the capacity to rescue the metal-inhibited OCTN1 function. The conclusions drawn on OCTN1 can be extended to other members of the SLC22 family and orthologous transporters in fish. Full article
(This article belongs to the Special Issue Transport of Nutrients and Ions Relevant to Human Pathophysiology)
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17 pages, 4618 KiB  
Article
Accumulation of Anthocyanin in the Aleurone of Barley Grains by Targeted Restoration of the MYC2 Gene
by Anastasiya A. Egorova, Tatyana E. Zykova, Christian W. Hertig, Iris Hoffie, Sergey V. Morozov, Elena I. Chernyak, Artem D. Rogachev, Anna M. Korotkova, Alexander V. Vikhorev, Gennady V. Vasiliev, Olesya Y. Shoeva, Jochen Kumlehn, Sophia V. Gerasimova and Elena K. Khlestkina
Int. J. Mol. Sci. 2024, 25(23), 12705; https://doi.org/10.3390/ijms252312705 - 26 Nov 2024
Cited by 1 | Viewed by 1715
Abstract
Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented [...] Read more.
Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins. Full article
(This article belongs to the Section Molecular Plant Sciences)
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22 pages, 9617 KiB  
Article
The Role of Aspergillus niger in Regulating Internal Browning Involves Flavonoid Biosynthesis and the Endophytic Fungal Community of Pineapple
by Fei Shen, Guang Wang and Shijiang Zhu
J. Fungi 2024, 10(11), 794; https://doi.org/10.3390/jof10110794 - 15 Nov 2024
Viewed by 1431
Abstract
Endophytic fungi are commonly used to control plant diseases, overcoming the drawbacks of chemical agents. The internal browning (IB) of postharvest pineapple fruit, a physiological disease, leads to quality losses and limits industrial development. This work investigated the relationship among the effects of [...] Read more.
Endophytic fungi are commonly used to control plant diseases, overcoming the drawbacks of chemical agents. The internal browning (IB) of postharvest pineapple fruit, a physiological disease, leads to quality losses and limits industrial development. This work investigated the relationship among the effects of Aspergillus niger (An) on IB controlling, flavonoid metabolism and the endophytic fungal community of pineapple through metabolomics, transcriptomics, microbiomics and microorganism mutagenesis technology. We obtained an endophyte An that can control the IB of pineapple and screened its mutant strain AnM, through chemical mutagenesis, that cannot control IB. The transcriptome of fungi showed that An and AnM were different in oxidative metabolism. Transcriptome and metabolome analyses of pineapple showed that An upregulated genes of flavonoid synthesis, including dihydroflavonol 4-reductase and flavonoid 3′-monooxygenase and increased the flavonoid content in pineapple fruit, i.e., Hispidulin, Hispidulin-7-O-Glucoside, and Diosmetin, while AnM could not. Microbiomics analysis identified an increase in the abundance of eight endophytic fungi in An-inoculated fruit, among which the abundance of six endophytic fungi (Filobasidium magnum, Naganishia albida, A. niger, Aureobasidium melanogenum, Kwoniella heveanensis and Lysurus cruciatus) was positively correlated with the content of three flavonoids mentioned above but not in AnM-inoculated fruit. Overall, this suggested, for the first time, that A. niger alleviated IB mainly by enhancing flavonoid synthesis and content and the abundance of endophytic fungi and by regulating the interaction between flavonoid content and endophytic fungi abundance in pineapple. This work adds to the understanding of the IB mechanism in postharvest pineapple and provides a new green approach for reducing postharvest losses and controlling physiological diseases. Full article
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11 pages, 735 KiB  
Article
Development and Large-Scale Production of High-Oleic Acid Oil by Fermentation of Microalgae
by Leon Parker, Kevin Ward, Thomas Pilarski, James Price, Paul Derkach, Mona Correa, Roberta Miller, Veronica Benites, Dino Athanasiadis, Bryce Doherty, Lucy Edy, Gawharah Alnozaili, Nina Reyes, Jon Wittenberg, Gener Eliares, Frédéric Destaillats, Walter Rakitsky and Scott Franklin
Fermentation 2024, 10(11), 566; https://doi.org/10.3390/fermentation10110566 - 6 Nov 2024
Cited by 3 | Viewed by 2534
Abstract
Our classical strain improvement began with an isolate showing 28% palmitic and 60% oleic acids. UV and chemical mutagenesis enhanced our strain’s productivity, carbon yield, and oleic acid content. The iterative methodology we used involved the creation of mutant libraries followed by clonal [...] Read more.
Our classical strain improvement began with an isolate showing 28% palmitic and 60% oleic acids. UV and chemical mutagenesis enhanced our strain’s productivity, carbon yield, and oleic acid content. The iterative methodology we used involved the creation of mutant libraries followed by clonal isolation, assessments of feedstock utilization and growth, oil titer, and the validation of oil composition. Screening these libraries facilitated the identification of isolates with the ability to produce elevated levels of oleic acid, aligning with the targets for high-oleic acid substitutes. Utilizing a classical strain improvement approach, we successfully isolated a high-oleic acid strain wherein the level of oleic acid was increased from 60 to >86% of total FA. The performance of the classically improved high oleic acid-producing strain was assessed at fermentation scales ranging from 1 L to 4000 L, demonstrating the utility of our strain and process at an industrial scale. These oils offer promise in various applications across both the food and industrial sectors, with the added potential of furthering sustainability and health-conscious initiatives. Full article
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16 pages, 2860 KiB  
Article
Computational Approach to Identifying New Chemical Entities as Elastase Inhibitors with Potential Antiaging Effects
by Giovanna Pitasi, Andrea Brancale, Sonia Floris, Antonella Fais, Rosaria Gitto and Laura De Luca
Int. J. Mol. Sci. 2024, 25(20), 11174; https://doi.org/10.3390/ijms252011174 - 17 Oct 2024
Cited by 3 | Viewed by 1728
Abstract
In the aging process, skin morphology might be affected by wrinkle formation due to the loss of elasticity and resilience of connective tissues linked to the cleavage of elastin by the enzymatic activity of elastase. Little information is available about the structural requirements [...] Read more.
In the aging process, skin morphology might be affected by wrinkle formation due to the loss of elasticity and resilience of connective tissues linked to the cleavage of elastin by the enzymatic activity of elastase. Little information is available about the structural requirements to efficiently inhibit elastase 1 (EC 3.4.21.36) expressed in skin keratinocytes. In this study, a structure-based approach led to the identification to the pharmacophoric hypotheses that described the main structural requirements for binding to porcine pancreatic elastase as a valuable tool for the development of skin therapeutic agents due to its similarity with human elastase 1. The obtained models were subsequently refined through the application of computational alanine-scanning mutagenesis to evaluate the effect of single residues on the binding affinity and protein stability; in turn, molecular dynamic simulations were carried out; these procedures led to a simplified model bearing few essential features, enabling a reliable collection of chemical features for their interactions with elastase. Then, a virtual screening campaign on the in-house library of synthetic compounds led to the identification of a nonpeptide-based inhibitor (IC50 = 60.4 µM) belonging to the class of N-substituted-1H-benzimidazol-2-yl]thio]acetamides, which might be further exploited to obtain more efficient ligands of elastase for therapeutic applications. Full article
(This article belongs to the Section Molecular Pharmacology)
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