Journal of Biological Engineering最新文献

{"title":"Enhanced enzyme activity and stability through immobilization of recombinant chitinase on sodium alginate-modified rice husk beads for efficient decolorization of synthetic dyes.","authors":"Shaimaa A Nour, Ebtehag A E Sakr, Heba Kandil","doi":"10.1186/s13036-025-00546-4","DOIUrl":"10.1186/s13036-025-00546-4","url":null,"abstract":"<p><strong>Background: </strong>The energy efficiency and environmental friendliness of recombinant chitinase A make it a promising candidate for industrial applications as a sustainable catalyst. For the first time, a very stable and an efficient biocatalyst was developed to decolorize synthetic dyes by immobilizing Serratia marcescens chitinase A (SmChiA) onto beads comprised of sodium alginate (SA) and modified rice husk powder (mRHP). The mRHP was produced by treating rice husk powder with citric acid, which was then combined with SA at three different concentrations (25, 50 and 100% of SA weight) and cross-linked with calcium chloride to form the beads. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide facilitates the formation of amide bonds that covalently bind SmChiA to the beads. The effectiveness of the synthesis and immobilization processes was confirmed using characterization methods (scanning electron microscopy, SEM and Fourier transform infrared spectroscopy, FTIR).</p><p><strong>Results: </strong>Beads with 50% mRHP and 1.75 UmL^- 1 of enzyme solution achieved the highest immobilization after 5 h of activation. The immobilized SmChiA demonstrated superior pH, temperature, and storage stability in respect to its free relative. The K_m value was 3.33 mg/mL, while the V_max was 4.32 U/mg protein/min. Activation energy (Ea), denaturation (E_d), half-lives (T_1/2), and decimal reduction time (D-values) were evaluated for immobilized and free SmChiA. The immobilization of SmChiA increased its affinity for the substrates by around 2.12 to 2.18 times. Compared to free chitinase, immobilized chitinase demonstrated greater durability after 22 reuses, maintaining its full activity. This proved the suitability of SA-mRHP beads as a cross-linker for chitinase immobilization. Crystal violet, malachite green, safranin, and methylene blue were more effectively decolorized from aqueous solutions by the immobilized SmChiA at a contact period of 84-h, dosage of 2.625 U/1.5 g, and temperature of 30 ^◦C. Using an immobilized biocatalyst, the biodegradation was also examined using UV, FTIR, and SEM-EDX. The results confirmed the dye degradation.</p><p><strong>Conclusion: </strong>A variety of dyes could be safely removed from the environment using our bioremediation procedures. To the best of our knowledge, no studies had been conducted on the application of immobilized chitinase for dye removal.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"78"},"PeriodicalIF":6.5,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376360/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simultaneous online monitoring of viscosity and oxygen transfer rate in shake flask cultures.","authors":"René Hanke, Michaela Sieben, Maurice Finger, Kilian Schnoor, Simon Jeßberger, Julia Weyand, Lluìs Coloma de la Fuente, Marcel Mann, Amizon Azizan, Udo Kosfeld, Jochen Büchs","doi":"10.1186/s13036-025-00552-6","DOIUrl":"10.1186/s13036-025-00552-6","url":null,"abstract":"<p><p>Shake flasks are among the most relevant culture vessels for early-stage process development of viscous microbial cultures. While online process monitoring systems are available for temperature, pH, biomass concentration, dissolved oxygen tension and respiration activity, online measuring techniques for viscosity are not yet commercially available. Especially during the production of biopolymers and the cultivation of filamentous fungi or bacteria, quantification of fermentation broth viscosity is essential to ensure adequate mixing as well as gas/liquid mass and heat transfer. In this work, a previously developed quantitative online viscosity measurement technique, termed ViMOS, is refined to monitor the apparent viscosity of up to eight shake flask cultures in parallel. In addition, the necessary preparation to ensure reproducible measurements is elucidated. By cultivating the two exopolysaccharide forming bacterial strains, Paenibacillus polymyxa and Xanthomonas campestris, as well as the filamentous fungus Trichoderma reesei, the ViMOS was successfully validated for viscosity values up to 120 mPa·s. The combination with oxygen transfer rate monitoring via a RAMOS device allowed to detect microbial growth phases, oxygen limitations, biopolymer production and degradation, as well as the morphological development of filamentous cultures. This dual online monitoring has the potential to improve screening conditions and simplify scale-up procedures of small-scale bioprocesses.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"77"},"PeriodicalIF":6.5,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12374283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced meropenem activity and stability following load in Polyvinyl alcohol nanofiber scaffolds with sitagliptin as quorum sensing inhibitor on Pseudomonas aeruginosa.","authors":"Abdelrahman A Mohsen, Taghrid S El-Mahdy, Mohamed Emara, Samar A Salim","doi":"10.1186/s13036-025-00549-1","DOIUrl":"10.1186/s13036-025-00549-1","url":null,"abstract":"<p><strong>Background: </strong>The increasing resistance of bacteria to conventional antibiotics poses a significant health challenge. Innovative strategies, such as combining antibiotics with agents like quorum sensing inhibitors (QSIs), have been developed to combat this issue. QSIs enhance antibiotic efficacy without inhibiting bacterial growth, minimizing the risk of resistance.</p><p><strong>Aims: </strong>Evaluate the combined effect of Sitagliptin (STG) as a QSI with Meropenem (MER), fabricate drug-loaded Polyvinyl Alcohol (PVA) nanofibers, and investigate their antimicrobial activity against standard Pseudomonas aeruginosa (PAO1) and carbapenem-resistant P. aeruginosa (CRPA).</p><p><strong>Methods: </strong>The combinatorial effect was assessed using a checkerboard assay. PVA/STG, PVA/MER, and PVA/STG/MER nanofibers were fabricated with varying concentrations of STG (2, 4, 8 mg/mL) and MER (5, 7, 9 mg/mL) via electrospinning. Characterization was performed using FTIR, XRD, and SEM techniques.</p><p><strong>Results: </strong>STG significantly reduced the minimum inhibitory concentration (MIC) of MER. The 1:2 STG to MER ratio exhibited the highest antimicrobial activity, achieving a comparable zone of inhibition to the highest concentration of MER while utilizing nearly half the amount of MER. The stability of the loaded scaffolds was maintained over three months at 2-8 °C.</p><p><strong>Conclusions: </strong>Our results underscore the successful fabrication of nanofiber scaffolds and the effectiveness of STG and MER-loaded nanofibers as promising wound dressings for cutaneous P. aeruginosa infections. This study highlights the potential of our innovative nanofiber system to enhance treatment efficacy against multidrug-resistant bacteria, offering a personalized and rapid response wound dressing solution for medical professionals. Ultimately, it shows promise to improve patient recovery and quality of life while minimizing systemic side effects.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"76"},"PeriodicalIF":6.5,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12366121/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144882866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Baculovirus-mediated production and purification of ferritin nanoparticles for rift valley fever vaccine development.","authors":"Margarida Q Rodrigues, Ashni Tambaclal, Brian Kloss, Paula M Alves, António Roldão","doi":"10.1186/s13036-025-00550-8","DOIUrl":"10.1186/s13036-025-00550-8","url":null,"abstract":"<p><strong>Background: </strong>Rift Valley fever (RVF) is a WHO-prioritized zoonotic, vector-borne disease with no licensed prophylaxis available for humans, highlighting the need for effective vaccine strategies. Nanoparticle-based platforms for antigen presentation offer a promising approach for vaccine development.</p><p><strong>Results: </strong>In this work, we engineered ferritin (Ft) nanoparticles to display the immunogenic Gn domain of RVF virus (GnFt) and systematically assessed the production, purification, and physico-chemical properties of the purified nanoparticles. Baculovirus-based expression systems were evaluated in insect (Sf9, High-Five™, Tnao38, and Tnms42) and mammalian cells (HEK293 and CHO), revealing Sf9 cells as the most efficient host for producing GnFt nanoparticles. In addition, affinity-based chromatography was explored, yielding GnFt nanoparticles of > 95% purity (as assessed by SDS-PAGE) and an overall production yield of 0.2 mg/L culture. Biophysical characterization (e.g., high-performance liquid chromatography, dynamic light scattering, electron microscopy, and mass photometry) confirmed proper 24-mer nanoparticle assembly (1,344 kDa and 20 nm) and structural integrity. Binding affinity to Gn-targeting monoclonal antibodies was demonstrated by biolayer interferometry, with dissociation constants in the nM range, indicating retained antigenic functionality.</p><p><strong>Conclusions: </strong>These findings demonstrate the successful development of a platform for producing structurally stable, pure, and functional Gn-presenting ferritin nanoparticles, supporting their potential use for RVF vaccine development.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"75"},"PeriodicalIF":6.5,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12355885/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing bone regeneration using kaempferol as an osteoprotective compound: signaling mechanisms, delivery strategies, and potential applications.","authors":"Mojdeh Salehi Namini, Sanam Mohandesnezhad, Sadaf Mohandesnezhad, Vahid Mansouri, Lobat Tayebi, Nima Beheshtizadeh","doi":"10.1186/s13036-025-00545-5","DOIUrl":"10.1186/s13036-025-00545-5","url":null,"abstract":"<p><p>Various plants, including fruits, vegetables, and spices, contain kaempferol, a bioflavonoid compound with diverse medicinal effects, such as antioxidant, antibacterial, and anti-inflammatory characteristics. Furthermore, this compound exhibits multiple health-promoting properties, including osteoprotection and osteogenesis, primarily by modulating various cell-signaling pathways. This review aims to illustrate the medical advantages of kaempferol and its role in regulating bone metabolism through cell signaling mechanisms. Numerous studies have demonstrated the bone-protective properties of kaempferol and its encapsulated form. Further research is needed to clarify the optimal dosages, toxicity, safety, and other potential mechanisms of action. This review demonstrates that several signaling pathways, including nuclear factor-kappa B (NF-κB), estrogen receptor, mitogen-activated protein kinase (MAPK), bone morphogenetic protein-2 (BMP-2), and mammalian target of rapamycin (mTOR) signaling pathways, regulate the osteogenesis and anti-osteoporotic effects of kaempferol as an osteoprotective compound. However, the main limitations to applying kaempferol in bone-related disorders are its low stability and absorption. One of the promising approaches to increasing its effectiveness is using delivery-related strategies such as encapsulation, scaffolding, hydrogels, and liposomes to constantly release kaempferol and subsequently enhance its bioavailability and absorption. Thus, this review has attempted to exhibit the understanding of the benefits of kaempferol as a new compound in regulating bone-related signaling pathways and various available delivery approaches to improve its therapeutic potential for treating bone-related diseases.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"74"},"PeriodicalIF":6.5,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12330109/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144794520","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volatile fatty acids recovery from thermophilic acidogenic fermentation using hydrophobic deep eutectic solvents.","authors":"Can Liu, Xueyao Zhang, Qi Qiao, Zhiwu Wang, Qing Shao, Jian Shi","doi":"10.1186/s13036-025-00544-6","DOIUrl":"10.1186/s13036-025-00544-6","url":null,"abstract":"<p><strong>Background: </strong>Volatile fatty acids (VFA) derived from acidogenic fermentation can be recovered as precursors for synthesizing value-added chemicals to replace those from fossil fuels. However, separating VFAs from the fermentation broth with complex constituents and a high-water content is an energy-intensive process.</p><p><strong>Results: </strong>This study developed an innovative membrane extraction technology, utilizing hydrophobic deep eutectic solvents (HDESs) as the acceptor phase along with an omniphobic membrane contactor for efficient extraction of anhydrous VFAs. All tested HDESs, three terpene-based type V HDESs and two tetraalkylammonium halide-based type III HDESs, were found to effectively extract VFAs at pH 3, with extraction recovery percentages (ERPs) up to 80% and 92% for 4 C- and 5 C- VFAs, respectively. However, the ERP of type V HDESs decreased significantly when the aqueous phase was adjusted to pH 6. Molecular simulations suggest that the VFA-HDES interactions vary with VFA dissociation, where the ion-dipole interactions between VFA conjugate bases and hydrogen bond donors at near-neutral pH conditions may destabilize the type V HDES structure and lead to reduced extraction efficiency. The temperature increases from 25 °C to 55 °C did not significantly impact VFA distribution, but a higher temperature could enhance cross-membrane mass transfer.</p><p><strong>Conclusions: </strong>This study demonstrated a novel continuous VFA extraction technology based on HDESs and elucidates the impact of temperature, pH, impurities in real fermentate and the applicability of an integrated membrane system through combined experimental and computational approaches.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"73"},"PeriodicalIF":6.5,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12315322/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144760118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Capture and detection of extracellular vesicles derived from human breast cancer cells using a 3D self-assembled nanostructured SiO₂ microfluidic chip.","authors":"Carolina Cabeza, Felipe Rojas, Lorena Lobos-González, Dominique Lemaitre, Juan Villena, María Luisa Cordero, Natalia Hassan, Rina Ortiz","doi":"10.1186/s13036-025-00512-0","DOIUrl":"10.1186/s13036-025-00512-0","url":null,"abstract":"<p><strong>Background: </strong>Tumor-derived extracellular vesicles offer a minimally invasive approach to evaluate tumor progression and metastasis. However, detecting biomarkers, such as extracellular vesicles in body fluids during the early stages of disease, remains a significant challenge. Conventional methods like ultracentrifugation-based isolation or Western blot protein quantification are time-consuming, require large sample volumes, and offer low yield and sensitivity. Therefore, the development of new biosensors for the specific and efficient analysis of tumor extracellular vesicles is urgently needed.</p><p><strong>Methods: </strong>Microfluidic devices provide extraordinary benefits for bioanalysis, offering a large surface area for the contact between target molecules and the biosensor, significantly enhancing the specificity, efficiency, and speed. These devices also enable nanoscale and microscale work using reduced sample volumes. In this study, we developed a three-dimensional self-assembled SiO₂-based nanostructured microfluidic chip, bioconjugated with specific antibodies targeting exosomal markers for the selective capture of CD63- and CD81-positive extracellular vesicles from breast cancer-derived conditioned cell culture media.</p><p><strong>Results: </strong>The three-dimensional SiO₂-based microfluidic chip effectively captured extracellular vesicles expressing CD63 and CD81 antigens from breast cancer cell culture media. This evidence demonstrates the potential of this platform to detect extracellular vesicles as biomarkers for cancer, providing a specific and efficient, non-invasive approach for cancer diagnostics.</p><p><strong>Conclusions: </strong>This study highlights the potential application of three-dimensional SiO₂-based microfluidic chips for detecting extracellular vesicles as a non-invasive liquid biopsy tool for breast cancer. The findings show a specific and efficient device as an alternative to conventional biomarker detection techniques.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"71"},"PeriodicalIF":6.5,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12305923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144742132","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fabrication of BMSCs-affinity peptide functionalized blended hyaluronic acid/polydopamine nanofibrous scaffolds to controlled Mg ion release and improved osteogenic differentiations for accelerating bone regeneration.","authors":"Jingzhe Zhang, Xinkun Wang, Xinbiao Fu, Ye Li","doi":"10.1186/s13036-025-00529-5","DOIUrl":"10.1186/s13036-025-00529-5","url":null,"abstract":"<p><p>Bone regeneration requires innovative solutions to enhance osteogenic differentiation and support effective tissue repair. This study presents a novel approach to bone tissue engineering by developing peptide-functionalized nanofibrous scaffolds (NFS). The fabrication of a blended hyaluronic acid (HA) and polydopamine (PD) scaffold functionalized with bone marrow mesenchymal stem cells (BMSCs)-affinity peptides (AP) designed to control magnesium ion (Mg) release, which supports BMSCs' osteogenic differentiation and bone regeneration. Characterization studies, including fourier-transform infrared spectroscopy (FTIR) and morphological analysis, confirmed the hydrophilic properties of HA/PDNFS@BMSCs-AP scaffolds, which enhance cell adhesion and proliferation. In vitro and In vivo assessments revealed that the scaffolds significantly promote osteogenesis through AP-induced pathways such as extracellular signal-regulated kinase pathway (ERK) and Phosphatidylinositol 3-kinase (Akt),. Animal model experiments demonstrated accelerated bone repair, supporting the potential of HA/PDNFS@BMSCs-AP scaffolds for targeted bone defect healing. These findings highlight the promise of functionalized nanofibrous scaffolds in bone tissue engineering and their potential application in regenerative medicine and translational research.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"72"},"PeriodicalIF":6.5,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12305988/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144742176","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to: Challenges and progress of neurodrug: bioactivities, production and delivery strategies of nerve growth factor protein.","authors":"Nan Zhou, TingWei Gu, Yang Xu, Yuda Liu, LiHua Peng","doi":"10.1186/s13036-025-00543-7","DOIUrl":"10.1186/s13036-025-00543-7","url":null,"abstract":"","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"70"},"PeriodicalIF":6.5,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12305953/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simultaneous multigene integration in Aspergillus fumigatus using CRISPR/Cas9 and endogenous counter-selectable markers.","authors":"Luis Enrique Sastré-Velásquez, Natalia Mach, Birte Mertens, Alexander Kühbacher, Petra Merschak, Alex Dallemulle, Lukas Lechner, Clara Baldin, George Diallinas, Fabio Gsaller","doi":"10.1186/s13036-025-00539-3","DOIUrl":"10.1186/s13036-025-00539-3","url":null,"abstract":"<p><strong>Background: </strong>The discovery of CRISPR/Cas9 and its subsequent accessibility in daily research initiated a new era in genome editing. This game-changing genetic instrument enabled a vast array of challenging applications requiring site-specific genome engineering as well as applications involving the equipment of cells with additional genetic traits. Despite the undisputed benefits of this technology, for facile and efficient selection of successfully manipulated cells selectable markers remain indispensable. Over the past years endogenous counter-selectable markers have come into focus in antifungal research enabling site-directed integration of multiple genes into the genome of the human mold pathogen Aspergillus fumigatus. However, gene cassettes had to be transformed in a consecutive manner keeping multigene integrations laborious and time-consuming.</p><p><strong>Results: </strong>In this work, we coupled the use of CRISPR/Cas9 with endogenous counter-selectable markers to achieve the simultaneous integration of multiple expression cassettes. The three markers used in this work included the herein employed azgA and the previously identified fcyB and cntA, responsible for 8-azaguanine, 5-fluorocytosine and 5-fluorouridine uptake, respectively. Exploiting their role in uptake of different selective agents, a triple selective transformation procedure and genomic integration of three expression cassettes in A. fumigatus was successfully accomplished. In addition to three distinct cellular reporters, we introduced strain-specific fluorescent reporters into four isolates displaying different levels of antifungal azole resistance to subsequently visualize and monitor their growth patterns in the same growth environment.</p><p><strong>Conclusions: </strong>The technology described in this study significantly streamlines the genetic manipulation process, reducing both time and labor associated with sequential transformations. By enabling the introduction of multiple genetic traits in a single transformation event, this strategy provides a flexible and efficient platform for a wide range of applications. As such, it enhances the potential for rapid and effective multigene integration, advancing the field of genetic engineering in fungi.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"69"},"PeriodicalIF":6.5,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12302872/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731106","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}