Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 Efficient in vivo Directed Evolution in E. coli using a Gibson Assembly-Adapted EvolvR System 65 72 1596 10.22104/mmb.2025.7751.1177 EN Yousef Vatanparast Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Gholamhossein Ebrahimipour Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Mohammad Yaghoubi-Avini Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 0000-0001-5761-1062 Journal Article 2025 07 18 Genetic diversity is vital for species adaptation and evolution, enhancing resilience to environmental changes and improving desirable traits. Directed evolution simulates natural selection in labs to engineer proteins and microorganisms, utilizing iterative cycles of genetic variation to achieve desirable characteristics. EvolvR is a system that can continuously diversify all nucleotides during an adjustable window at user-defined locations. It is achieved by mutagenesis using engineered DNA polymerases directed to the target site via CRISPR-directed nickases. Although the typical plasmid assembly and gRNA insertion method is Golden Gate cloning, the aim of this study was to set up EvolvR according to our equipment and conditions. The gRNA targeting rpsE in E. coli DH5α was selected from a previous study. Specific primers that included the gRNA sequence and provided homology on one side were inserted into the EvolvR plasmid using the Gibson assembly method. The constructed plasmid was chemically transformed into E. coli DH5α. Bacterial resistance was evaluated by colony counting on culture media containing 50, 100, and 500 µl/ml of spectinomycin. Results showed that the number of E. coli DH5α cells in an antibiotic-free medium was 11×108 CFU.mL-1, while no growth was observed at any antibiotic concentration. The non-induced EvolvR cells did not grow in a medium containing 100 and 500 μg.mL-1 spectinomycin, but grew at 20×106 CFU.mL-1 in a medium with 50 μg.mL-1 antibiotic. Induction of the EvolvR system resulted in a dramatic increase in spectinomycin-resistant mutants, yielding up to 4×108 CFU.mL-1 on 100 μg⋅mL−1 spectinomycin and a resistance frequency order of magnitude higher than previously reported. Our findings validate Gibson assembly as a robust and accessible alternative to Golden Gate for constructing EvolvR systems and emphasize the high efficacy achievable with a strategically targeted single gRNA.

https://mmb.irost.ir/article_1596_c9d44f25340a0b5a4163ef430842b606.pdf

Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 Anticoagulant Activity of Exopolysaccharide Extracted from Two Native Microalgae 73 80 1609 10.22104/mmb.2025.7805.1182 EN Zahra Mousavian Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P. O. Box 3353-5111, Tehran, Iran Maliheh Safavi Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P. O. Box 3353-5111, Tehran, Iran Farzaneh Azizmohseni Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P. O. Box 3353-5111, Tehran, Iran Saeed Mirdamadi Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P. O. Box 3353-5111, Tehran, Iran Journal Article 2025 08 05 The increasing demand for natural substances in medicinal applications has made microalgae an attractive bioresource. The potential health benefits of polysaccharides from a variety of microalgae species have garnered attention. The aim of this study was to investigate and characterize the growth, biomass, and exopolysaccharide production profile of two microalgal strains, Chlorella sp. and Chlorella sorokiniana. The anticoagulant activities of extracted sulfated polysaccharides were evaluated using activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays. Higher carbohydrate production yield (19 (w/w)) was observed in Chlorella sorokiniana. Therefore, Chlorella sorokiniana can be used to produce biomass that is high in carbohydrates. The sulfated polysaccharide extracted from Chlorella sorokiniana and Chlorella sp. showed anticoagulant properties. C. sorokiniana and Chlorella sp. (strain D1) sPS were found to be less active than heparin in the measurement of aPTT and PT. To possess the same effect as heparin, greater polysaccharide concentrations were necessary These results demonstrate the potential of sulfated polysaccharides derived from microalgae to be used in the development of new therapeutic agents. The development of next-generation antithrombotic medications, which will have fewer side effects and a greater range of uses, may be facilitated by such studies.These results demonstrate the potential of sulfated polysaccharides derived from microalgae to be used in the development of new therapeutic agents. The development of next-generation antithrombotic medications, which will have fewer side effects and a greater range of uses, may be facilitated by such studies. Keywords: Microalgae, cultivation, exopolysaccharides, food supplement

https://mmb.irost.ir/article_1609_8c9301394b6d35c5dd2325b1a15143d8.pdf

Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 Polymicrobial Shifts in the Culturable Bacterial Microbiome Associated with Persian Oak Decline in Western Iran 81 94 1620 10.22104/mmb.2025.7777.1181 EN Elahe Ahmadi Department of Silviculture and Forest Ecology, Gorgan University of Agricultural Science and Natural Resources, Basij Square, P.O. Box: 4918943464, Gorgan, Iran Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran Mojegan Kowsari Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 0000-0002-6858-4052 Davood Azadfar Department of Silviculture and Forest Ecology, Gorgan University of Agricultural Science and Natural Resources, Basij Square, P.O. Box: 4918943464, Gorgan, Iran Gholamreza Salehi Jouzani Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran Journal Article 2025 08 04 Persian oak decline is a distinct syndrome within the broader oak decline complex observed in Iran, marked by excessive stem bleeding and larval galleries formed by the native buprestid beetle Agrilus hastulifer. To investigate this phenomenon, a comparative study was conducted on healthy and symptomatic trees across eight sites in Ilam province, western Iran. Culturable bacterial communities were identified using 16S rDNA sequencing. Symptomatic tissues from trees at Disease Index 5 yielded bacterial growth in 83.78% of samples—significantly higher than those from less affected trees. Bulk soil and rhizosphere samples also yielded greater bacterial yields than root, leaf, or stem tissues. Although bacterial community composition varied by site, diseased tissues consistently showed dominance of Enterobacteriaceae, while Bacillaceae and Moraxellaceae were more prevalent in healthy trees. Specific bacterial species, Brenneria goodwinii, Serratia marcescens, and Brenneria roseae, were strongly associated with diseased tissues, suggesting that necrosis was not due to random colonization. Campylobacter jejuni and an unidentified Clostridium taxon were frequently isolated from both healthy and diseased trees. These findings indicate a clear shift in the microbiome of diseased trees, with Enterobacteriaceae absent in healthy tissues. Crucially, no single dominant pathogen was identified, supporting the hypothesis that Persian oak decline is driven by a polymicrobial infection.

https://mmb.irost.ir/article_1620_30084e4009e9af0fa7af67e633ebbcd1.pdf

Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 Theoretical and Experimental Study of a Glycan-Coated Biosensor for Staphylococcus aureus Detection 95 105 1627 10.22104/mmb.2025.7874.1184 EN Vahid Reza Nafisi Department of Electrical Engineering and Information Technology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran Ensieh Fahimi Kashani Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran Zahra Mousavian Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran Yasamin Bide Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran Alireza Balaei Department of Electrical Engineering and Information Technology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran Journal Article 2025 09 15 Methicillin‑resistant Staphylococcus aureus is one of the community-associated pathogens with a high capacity for attachment to human tissues and biofilm formation. Therefore, the design of tools for rapid identification of this bacterium in environments and providing timely safety alerts is essential for public health. The aim of the present study was to identify a suitable coating that can be immobilized on an electrical sensor and effectively capture Staphylococcus cells on the sensor surface. The glycan Asialoganglioside GM2 (GA2) was selected as a candidate for this purpose, and molecular docking simulations were used to investigate and validate its potential to bind Staphylococcus receptors. The results showed that the interaction of GA2 with S. aureus receptors exhibited high binding affinity and relatively low RMSD values, indicating a stable and reliable binding interaction. This strong binding of GA2 with ClfB receptor suggests potential applications for GA2 in inhibiting bacterial adhesion and in developing diagnostic or therapeutic strategies targeting S. aureus. Also, this simulation finding was confirmed experimentally. A GA2 coating was applied to the sensor, increasing the probability of bacterial attachment to the sensor surface by more than eightfold. Also, the coating’s specificity to S. aureus detection was examined with E. coli (experimentally and theoretically). These findings may provide a basis for designing targeted therapeutics and diagnostics against this important pathogen.

https://mmb.irost.ir/article_1627_4f8c6782149e4a84a044a5c1eeb81032.pdf

Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 In silico prediction of the three-dimensional structure of the antimicrobial peptide Fa-AMP1 using a multi-tool approach and peptide-ligand docking 106 120 1628 10.22104/mmb.2025.7898.1186 EN Sahba Toosi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad. Mashhad. Iran. Farhad Shokouhifar Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad. Mashhad. Iran. Saeid Malekzadeh Shafaroudi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad. Mashhad. Iran. Mojtaba Mamarabadi Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad. Mashhad. Iran. Nasrin Moshtaghi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad. Mashhad. Iran. Journal Article 2025 09 30 The Fa-AMP1 peptide from buckwheat (Fagopyrum esculentum Moench) seed germ and 40 amino acid long has previously been reported to exhibit potent antifungal and antibacterial effects. The availability of the three-dimensional structure of this peptide could be very useful in investigating its antimicrobial function, but crystallographic information of this peptide is not available so far. The aim of the present study was to determine the three-dimensional structure of the Fa-AMP1 peptide using a comprehensive approach so that advanced modeling tools such as AlphaFold, SwissModel, and D-I-TASSER were predicted and evaluated. The predicted models were compared using validation tools QMEAN, Verify3D, WHATCHEC. The geometric position of amino acids in all three models was examined by drawing Ramachandran plots. The distance of all three models from each other was compared using the RMSD index, and the number and position of alpha-beta-coil folds were also compared as well as the position of disulfide bonds. Overall, the results showed that, the models presented by the AlphaFold and SwissModel tools have high convergence with each other, and the model presented by the D-I-TASSER tool also showed high similarity with the other two models, but did not show the necessary accuracy in the configuration of cysteine amino acids and the formation of expected disulfide bonds. In the second part of the article, the Swiss docking tool was used to investigate the interaction between the model predicted by alpha-fold and chitin as a target ligand which sourced from fungal cell wall. The docking results were analyzed based on the position of amino acids involved in binding to the ligand in the five complexes runs with the peptide model. The docking results showed that the model presented by AlphaFold in the fourth run of the results presented by Swiss docking can be placed at an appropriate distance from each other with the amino acids related to the binding site in the Fa-AMP1 peptide and the four amino acids form hydrogen bonds with the NAG1 and 2 monomers. In future studies, this prediction can be investigated by conducting additional molecular dynamics (MD) simulations.

https://mmb.irost.ir/article_1628_f3fb60a7c4ad1e1bb5fb45219e097edf.pdf

Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 The Emerging Role of Engineered Probiotics in Gastrointestinal Health: Mechanisms, Challenges, and Future Perspectives 121 130 1629 10.22104/mmb.2026.7952.1188 EN Kimiya Khani-Chegeni Department of Biology, Khor.C., Islamic Azad University, Khorramabad, Iran. Samaneh Sedighi-Khavidak Department of Biology, Ashk.C., Islamic Azad University, Ashkezar, Iran. and Medical Biotechnology Research Center, Ashk.C., Islamic Azad University, Ashkezar, Iran. Mohammad Rabbani Khorasgani Department of Biology, University of Isfahan, P.O. Box:81746-73441, Isfahan, Iran Farnaz Mozayani School of Medicine, Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran. Journal Article 2025 10 25 Traditional probiotics, such as Lactobacillus, Lactococcus, and Bifidobacteria, defined as live microorganisms used to manage gastrointestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), have demonstrated variable clinical efficacy. The advent of synthetic biology has ushered in a paradigm shift, enabling the rational design and production of Next-Generation Probiotics (NGPs) or engineered strains with precise therapeutic and diagnostic functionalities at targeted gastrointestinal sites. This review discusses predominant molecular mechanisms employed by engineered probiotics, including enhanced biosynthesis of bioactive metabolites like short-chain fatty acids (SCFAs) and site-specific delivery of immunomodulatory cytokines such as interleukin-10 (IL-10). Additionally, critical biosafety considerations related to biocontainment and horizontal gene transfer )HGT) are evaluated, highlighting advanced genetic safeguards such as CRISPR-Cas–based kill switches to mitigate dissemination risks. Regulatory frameworks governing these live biotherapeutic products (LBPs) are also analyzed. Future directions encompass the integration of state-of-the-art gene editing technologies and GI biosensors for in vivo disease monitoring, as well as the potential for engineered probiotics in personalized medicine. Collectively, advancements in synthetic biology and genomics have propelled engineered probiotics as programmable live therapeutics, offering novel avenues for the prevention, diagnosis, and treatment of refractory gastrointestinal diseases. These approaches, while navigating biosafety and regulatory challenges, promise enhanced clinical efficacy and pave the way for precision microbiome interventions.

https://mmb.irost.ir/article_1629_ba3abf3db77980ea1e9196e54ec7d7e8.pdf