Journal of biotechnology最新文献

{"title":"Engineering of endogenous plasmids in probiotic Escherichia coli Nissle 1917 for autonomous accumulation of 5‑aminolevulinic acid","authors":"Dong Yu ,&nbsp;Lin Zhu ,&nbsp;Ya-Xue Ding ,&nbsp;Li-Jing Mao ,&nbsp;Min Xiong ,&nbsp;Xiao-Xuan Jin ,&nbsp;Yujie Ma ,&nbsp;Feng-Qing Wang ,&nbsp;Liang-Bin Xiong","doi":"10.1016/j.jbiotec.2025.11.006","DOIUrl":"10.1016/j.jbiotec.2025.11.006","url":null,"abstract":"<div><div>5-aminolevulinic acid (ALA), a non‑proteinogenic δ‑amino acid, is a versatile compound with applications as a tumor-sensitizing agent in photodynamic therapy and a plant biostimulant that enhances stress tolerance and photosynthetic efficiency. This study aimed to leverage the endogenous cryptic plasmids pMUT1 and pMUT2 of the probiotic <em>Escherichia coli</em> Nissle 1917 (EcN) to construct a self-sufficient strain with autonomous ALA biosynthesis capability, eliminating dependencies on exogenous plasmids or chemical inducers. A synthetic operon was designed on a re-engineered cryptic plasmid to express core genes of the C5 pathway (<em>gltX</em>, <em>hemA</em>, and <em>hemL</em>). Systematic evaluation demonstrated that overexpression of <em>gltX</em> alone resulted in minimal ALA accumulation (6.8 mg/L). In contrast, coordinated co-expression of <em>hemA</em> and <em>hemL</em> significantly increased ALA titers to 854.5 mg/L, highlighting their synergistic role in channeling carbon flux through the C5 pathway in EcN. Subsequent optimization of the <em>hemA‑hemL</em> cassette further elevated production to 925.1 mg/L. Notably, this enhanced ALA synthesis perturbed the cellular NADP⁺ /NADPH balance. To address this, we integrated <em>pos5</em>, encoding a NADP⁺‑dependent transhydrogenase, into the endogenous plasmid, enabling <em>in situ</em> NADPH regeneration and boosting ALA titers to 1306.8 mg/L. Scale up to a 5-L bioreactor with fed-batch cultivation and controlled glycerol feeding achieved an ALA titer of 3372.5 ± 162.7 mg/L at 108 h. This endogenous plasmid-centric approach establishes an inducer-free, antibiotic-independent microbial cell factory, positioning EcN as a universal platform for ALA production with potential in biomedical and agricultural applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 157-164"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Morphology and bioproduction potential of DCB-induced cell wall-deficient tobacco BY-2 cells and transcriptomic analysis","authors":"Uddhab Karki ,&nbsp;Shekoofeh Sadravi ,&nbsp;Katelin Kellar ,&nbsp;Marin Hirata ,&nbsp;Fnu Mamta ,&nbsp;Jianfeng Xu","doi":"10.1016/j.jbiotec.2025.11.007","DOIUrl":"10.1016/j.jbiotec.2025.11.007","url":null,"abstract":"<div><div>Plant cell cultures, especially tobacco BY-2 cells, represent a promising platform for recombinant protein production. However, their full potential is hindered by several challenges. Some of these challenges, such as inefficient protein secretion, the presence of large vacuoles, and difficulties in cryopreservation can be attributed to the rigidity of the plant cell wall. We developed and characterized cell wall-deficient BY-2 cells by gradually adapting them to the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB). The DCB-adapted cells exhibited marked morphological changes, including cell clumping due to pectin overaccumulation and an ∼80 % reduction in cellulose content. Despite these alterations, the DCB-adapted BY-2 cells maintained robust growth and demonstrated efficient <em>Agrobacterium</em>-mediated transformation. The production and secretion levels of the model protein EGFP and the therapeutic protein human EPO were 2.1- to 2.8-fold higher than those observed in native BY-2 cells. However, secretion of EGFP fused with a hydroxyproline-<em>O</em>-glycosylated module (SP)₃₂ was dramatically enhanced by up to 35-fold. Transcriptomic analysis revealed broad gene expression changes, including down-regulation of genes involved in cellulose biosynthesis and upregulation of genes associated with branched pectic polysaccharide synthesis. These results provide a proof of concept that cell wall-deficient plant cell lines can serve as effective platforms for recombinant protein production, laying foundation for further engineering of plant cells as next-generation biofactories.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 170-181"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471132","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient recombinant protein secretion in Mycobacterium smegmatis: A valuable platform for protein production involved in biomedical development targeting mycobacterial diseases","authors":"Hiroto Takeuchi ,&nbsp;Satoru Mizuno ,&nbsp;Kazuhiro Matsuo","doi":"10.1016/j.jbiotec.2025.10.013","DOIUrl":"10.1016/j.jbiotec.2025.10.013","url":null,"abstract":"<div><div><em>Mycobacterium smegmatis</em> is often used as a host for preparing various recombinant mycobacterial proteins that are used for structural and functional studies. The advantages of <em>M. smegmatis</em> as a host are that it is fast-growing, non-pathogenic, easy to manipulate, and has similar post-translational modifications to other mycobacteria. It is critical to express recombinant proteins with similar conformations and/or functions to native proteins from major pathogenic mycobacterial strains, including both <em>Mycobacterium tuberculosis</em> and non-tuberculosis mycobacteria. Accordingly, <em>M. smegmatis</em> has been chosen as a model for both systems. Therefore, the <em>M. smegmatis</em> expression platform must be applicable not only for functional studies but also for studies of antigen production. In this study, we examined whether the secretion system of <em>M. smegmatis</em> was suitable for establishing a vector capable of producing large amounts of recombinant protein. To do so, we examined the secretions of several mycobacterial secretory proteins in <em>M. smegmatis</em> to identify an effective signal sequence for efficient production. We found that a signal peptide from the <em>Mycobacterium kansasii</em> homolog of Rv1926c demonstrated efficient secretion of targeted mycobacterial proteins. Next, mutant signal peptide analysis revealed that the length of the hydrophobic amino acid stretch region plays a critical factor during signal peptide function. Overall, our data show that the secretion system of <em>M. smegmatis</em> may have potential for production of various mycobacterial proteins for vaccine development and diagnostic research.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 148-156"},"PeriodicalIF":3.9,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145437810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic engineering of Cupriavidus necator using sucrose phosphorylase pathway for polyhydroxybutyrate production from sucrose.","authors":"Vijaykumar Khonde, Mandar Deshpande, Dheeraj Mahajan, Meenakshi Tellis, Pramod Kumbhar, Anand Ghosalkar","doi":"10.1016/j.jbiotec.2025.08.002","DOIUrl":"10.1016/j.jbiotec.2025.08.002","url":null,"abstract":"<p><p>Sucrose-rich feedstocks are the most suitable raw materials for the production of biodegradable polymers like Polyhydroxyalkanoates (PHAs). Cupriavidus necator, a versatile microorganism with natural ability to accumulate poly(3-hydroxybutyrate) (PHB), has been shown to utilize a diverse set of carbon sources including sugars, oils, and gaseous feedstock like CO₂. However, both wild-type and mutant strains of C. necator cannot metabolize sucrose, limiting its utility in industrial production using sucrose-rich feedstocks. We developed metabolically engineered strains of C. necator for sucrose utilization by introducing sucrose phosphorylase pathway. Among all the recombinant strains, C. necator harbouring sucrose phosphorylase from Rhizobium vitis (CN-SPrv) along with sucrose permease and phosphoglucomutase from Escherichia coli demonstrated the most efficient utilization of sucrose. The CN-SPrv strain was evaluated for the utilization of sucrose using cane biosyrup and resulted in 60 g/L of PHB titer and 31 % yield on a consumed sugar basis in fed-batch mode of fermentation. This is the first report on metabolic engineering of C. necator using the sucrose phosphorylase pathway for PHB production.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"22-30"},"PeriodicalIF":3.9,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144794555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synergistic antitumor effects of recombinant Interleukin-21 and 5-fluorouracil: A novel therapeutic approach against hepatocellular carcinoma","authors":"Mina Yazdi,&nbsp;Zahra Hajihassan","doi":"10.1016/j.jbiotec.2025.10.012","DOIUrl":"10.1016/j.jbiotec.2025.10.012","url":null,"abstract":"<div><div>Interleukin-21 (IL-21) is a pleiotropic cytokine that enhances both humoral and cellular immunity by increasing B-cell proliferation, T-cell effector function, and natural killer (NK) cell cytotoxicity. Studies have demonstrated its potential as an anti-cancer agent in the treatment of melanoma, neuroblastoma, fibrosarcoma, hepatocellular carcinoma (HCC) and non-small-cell lung carcinoma. The aim of this study was to evaluate the cytotoxic effect of recombinant human IL-21 (rhIL-21) protein, both alone and in combination with 5-fluorouracil, a potent anticancer agent, on the HepG2 cell line. To achieve this, the protein was synthesized in the periplasmic space of <em>Escherichia coli</em> and subsequently purified. The MTT viability assay results demonstrated that treating HepG2 cells with rhIL-21 alone resulted in 54.81 % and 59.75 % cytotoxicity after 24 and 48 h respectively, with corresponding IC50 values of 108 ng/mL and 68.90 ng/mL. Furthermore, the level of cytotoxicity remained constant at concentrations above 150 ng/mL of rhIL-21. Treatment with 25 µg/mL of 5-fluorouracil alone resulted in 21.09 % and 27.71 % cytotoxicity after 24 and 48 h, respectively. However, combining 5-fluorouracil with 150 ng/mL of rhIL-21 significantly increased toxicity, reaching 81.15 % and 89.34 % after 24 and 48 h, respectively — an improvement of approximately 60 %. This underscores the synergistic potential of rhIL-21 in combination therapy, with IC50 values of 25 µg/mL of 5-fluorouracil and 30.96 or 15.79 ng/mL of rhIL-21 after 24 or 48 h, respectively. This regimen demonstrated strong cytotoxic effects against HepG2 cells while showing low toxicity towards normal L929 fibroblasts, suggesting its potential safety and therapeutic applicability.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 137-147"},"PeriodicalIF":3.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hairy roots as a biotechnological tool for medicinal plant secondary metabolites: A systematic review","authors":"Amine Elbouzidi ,&nbsp;Mohamed Taibi ,&nbsp;Mounir Haddou ,&nbsp;Abdellah Baraich ,&nbsp;Ibrahim Sadougui ,&nbsp;Reda Bellaouchi ,&nbsp;Abdeslam Asehraou ,&nbsp;Khalid Chaabane ,&nbsp;Bouchra El Guerrouj ,&nbsp;Mohamed Addi","doi":"10.1016/j.jbiotec.2025.10.010","DOIUrl":"10.1016/j.jbiotec.2025.10.010","url":null,"abstract":"<div><div>Hairy root cultures, generated via Agrobacterium rhizogenes-mediated transformation, are a major advance in plant biotechnology. They exhibit genetic stability, autonomous growth without exogenous phytohormones, and sustained high-yield production of bioactive secondary metabolites. These systems have applications in pharmaceuticals, nutraceuticals, and cosmetics. Notable metabolites include vincristine from <em>Catharanthus roseus</em> (L.) G. Don, withanolides from <em>Withania somnifera</em> (L.) Dunal, and ginsenosides from <em>Panax ginseng</em> C.A. Meyer, displaying anticancer, anti-inflammatory, antimicrobial, and neuroprotective activities. Despite these advantages, challenges such as suboptimal yields in certain species, inefficient transformation protocols, and complex regulatory frameworks limit industrial adoption. To address this, a systematic review was performed following PRISMA guidelines. Data were retrieved from PubMed, Scopus, and Web of Science using terms including “hairy root culture,” “secondary metabolite biosynthesis,” and “elicitation strategies.” The review included experimental studies on medicinal plant species capable of metabolite production via hairy roots, excluding theoretical studies and non-medicinal plants. The review pursued five primary objectives: (1) to compile a comprehensive inventory of medicinal plant species utilized in hairy root research; (2) to critically evaluate elicitation methods for enhancing metabolite production; (3) to examine current challenges related to scale-up of hairy root cultures for industrial application; (4) to identify actionable strategies to overcome existing limitations; and (5) to highlight the pharmaceutical properties of secondary metabolites derived from hairy roots. Empirical findings indicate that elicitors—such as jasmonic acid, salicylic acid, and emerging nanomaterials—significantly enhance metabolite accumulation. Molecular tools further optimize biosynthetic pathways. Nevertheless, species-specific constraints and unharmonized regulatory guidelines continue to impede commercialization. Integration of genetic engineering, bioprocess optimization, and regulatory science is essential to fully exploit the biotechnological potential of hairy root culture systems for pharmaceutical development.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 117-136"},"PeriodicalIF":3.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of benzene-ethanol extractives on deep eutectic solvent-mediated lignin extraction from poplar and eucalyptus","authors":"Rongxuan Li,&nbsp;Shubin Wu","doi":"10.1016/j.jbiotec.2025.10.011","DOIUrl":"10.1016/j.jbiotec.2025.10.011","url":null,"abstract":"<div><div>Deep eutectic solvents (DES), an emerging class of green solvents, have demonstrated remarkable efficiency in the fractionation of lignocellulosic biomass. Benzene-ethanol extractives in woody biomass significantly affect the efficiency of DES-mediated fractionation. Therefore, investigating influence of benzene-ethanol extractives on DES-mediated pre-treatment is essential for optimizing of poplar and eucalyptus fractionation. Removing benzene-ethanol extractives significantly increased the polar component of the surface free energy (SFE) in both poplar and eucalyptus biomass. This prominently enhanced the wettability of DES on the raw materials, thereby ameliorating heat and mass transfer effectiveness. Furthermore, the removal of these extractives was instrumental in modifying the surface pore structure of the raw materials, thereby facilitating a further meliorative fractionation effect of DES. Following the removal of the benzene-ethanol extract, the delignification efficiency for poplar and eucalyptus significantly increased to 81.92 % and 80.16 %, respectively. Concurrently, the extracted lignin exhibited higher hydroxyl content, a more complex aromatic ring structure, and improved thermal stability. All these findings substantiate that the removal of benzene-ethanol extractives represents a promising strategy for enhanced DES fractionation efficiency.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 107-116"},"PeriodicalIF":3.9,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145400799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Semi-rational design of the substrate-binding pocket of acyl-CoA dehydrogenase Tfu_1647 to improve catalytic activity for 6-carbon substrates","authors":"Lixia Liu ,&nbsp;Shenghu Zhou ,&nbsp;Yu Deng","doi":"10.1016/j.jbiotec.2025.10.007","DOIUrl":"10.1016/j.jbiotec.2025.10.007","url":null,"abstract":"<div><div><em>Tfu</em>_1647, an acyl-CoA dehydrogenase, reversible catalyses the conversion of enoyl-CoA to acyl-CoA and is often utilized in the biosynthesis of adipic acid. However, its applicability constrained by a lack of three-dimensional structural information and its broad substrate acceptance. In this study, we demonstrate structural and biochemical investigations of 6-carbon substrate (6C-CoA) dehydrogenase <em>Tfu</em>_1647 with high-efficiency. Notably, <em>Tfu</em>_1647 displayed a high substrate affinity for the intermediate-length acyl-CoA, which is an essential precursor for the synthesis of fatty acids. A semi-rational design approach, informed by the crystal structure, was employed to enhance the specific activity of 6C-CoA by identifying effective variants. Within this cohort of variants, <em>Tfu</em>_1647^{Thr370Gln(T370Q)} demonstrated the highest affinity for 6C-CoA (<em>K</em>_d = 1.48 × 10⁻⁶ M⁻¹) and the greatest enzyme activity (<em>k</em>_cat = 0.98 min⁻¹), representing a 25.7 % improvement in binding affinity and a 2.3-fold increase in catalytic rate compared to wild-type <em>Tfu</em>_1647. A comprehensive comparison revealed that the <em>Tfu</em>_1647^T370Q enlarges the substrate pocket and subtly alters local electrostatics, thereby preserving FAD binding while improving substrate affinity (25.7 % lower <em>K</em>_d) and catalytic efficiency (3.7-fold higher <em>k</em>_cat/<em>K</em>_M) toward 6C-CoA. Molecular dynamics and free energy analyses further showed that this substitution refines substrate specificity, conferring a distinct preference for medium-chain (C6) substrates over shorter or longer acyl-CoAs. This finding paves the way for the rational design of <em>Tfu</em>_1647 for its application in adipic acid biosynthesis.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 96-106"},"PeriodicalIF":3.9,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145400788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rational comparison of biohydrogen production using Clostridium species through dark fermentation during anaerobic batch processes","authors":"Ludovic Vauthier,&nbsp;Emmanuel Rondags,&nbsp;Céline Loubière,&nbsp;Nakry Pen,&nbsp;Xavier Framboisier,&nbsp;Emmanuel Guedon,&nbsp;Stéphane Delaunay","doi":"10.1016/j.jbiotec.2025.10.009","DOIUrl":"10.1016/j.jbiotec.2025.10.009","url":null,"abstract":"<div><div>Bacteria of the genus <em>Clostridium</em> are remarkable for their ability to produce hydrogen from a wide range of substrates through dark fermentation. This type of fermentation has been extensively studied in the literature. However, a large number of culture conditions were applied, making it difficult to compare different strains in terms of hydrogen production performance. The potential of <em>Clostridium</em> is therefore not fully explored. This study compared the performances of four clostridial strains in terms of hydrogen production under batch conditions, in a stirred tank with a regulated pH, at atmospheric pressure. Glucose was used as the sole carbon substrate. <em>Clostridium pasteurianum</em> ATCC 6013 (= DSM 525) was the strain with the highest gas volume production of 866 (± 291) mL_gas/(L_bioreactor·h). This strain is able to exhibit a hydrogen to substrate yield of 1.73 (± 0.12) mol/mol and a hydrogen to carbon dioxide ratio of 1.03 (± 0.13) mol/mol, which are values comparable to those of other strains studied. By combining every parameter, it appears that ATCC 6013 is a certain strain of interest for high-volume H₂ production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 85-95"},"PeriodicalIF":3.9,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367715","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Construction of high protein-producing mutant yeast strains via point and structural mutageneses and their use for carotenoid production","authors":"Ryosuke Yamada ,&nbsp;Yoshifumi Inoue,&nbsp;Yukino Karitani,&nbsp;Rumi Sakaguchi,&nbsp;Takuya Matsumoto,&nbsp;Hiroyasu Ogino","doi":"10.1016/j.jbiotec.2025.10.008","DOIUrl":"10.1016/j.jbiotec.2025.10.008","url":null,"abstract":"<div><div>The yeast <em>Saccharomyces cerevisiae</em> is a safe microorganism with established industrial-scale culture techniques. Standard laboratory <em>S. cerevisiae</em> strains, such as the representative YPH499, are valuable hosts for producing proteins and chemicals through metabolic engineering. Consequently, there's a high demand for platform strains of <em>S. cerevisiae</em> with enhanced protein production capacity. We have previously established an efficient and straightforward technique for introducing point and structural mutations into yeast via plasmid introduction, leading to the generation of mutant strains with superior phenotypes. In this study, we aimed to develop <em>S. cerevisiae</em> mutants with high protein production capacity using techniques to introduce point and structural mutations. We introduced these mutations into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP). Since GFP is easily detected by its fluorescence, we selected mutants based on their fluorescence intensity. Consequently, YPH499/pEUPGGFP/Mu10G39, with a GFP fluorescence intensity 2.5-fold higher than that of the parent strain, was successfully obtained. Then, a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids, which was 2.9-fold higher than that produced by the parent strain. Transcriptome analysis suggested that YPH499Mu10G39 exhibited improved energy production, amino acid precursor supply, ribosome function, and stress tolerance, which may have contributed to its high protein production. In conclusion, by introducing point and structural mutations, we successfully developed the platform strain, YPH499Mu10G39, which is useful for the high production of various proteins. In the future, various proteins and useful chemicals can be produced through metabolic engineering using YPH499Mu10G39 as a platform strain.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 77-84"},"PeriodicalIF":3.9,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145359795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}