Biotechnology Journal最新文献

{"title":"The SpyTag/SpyCatcher System: Precise Regulation of Covalent Conjugation and Expansion of Application Scenarios","authors":"Meng-meng Cai,&nbsp;Zi-fu Ni,&nbsp;Zhong-ke Sun,&nbsp;Xiao-long Li,&nbsp;Yan-li Qi,&nbsp;Le Wang,&nbsp;Chao He,&nbsp;Cheng-wei Li","doi":"10.1002/biot.70131","DOIUrl":"10.1002/biot.70131","url":null,"abstract":"<div>\u0000 \u0000 <p>The SpyTag/SpyCatcher system is a modular protein assembly tool. Its core mechanism is the formation of isopeptide bonds, which achieves protein assembly through covalent coupling. This system is characterized by mild reaction conditions, rapid connection and no need for additional reagents, and shows good application potential in fields such as enzyme engineering. Although the system has made progress in application, it still faces challenges such as industrial scale and clinical immunogenicity. This paper systematically reviews the principle of the SpyTag/SpyCatcher system and its development progress. It also analyzes the deficiencies of the system in industrial applications, focuses on elaborating its specific application examples in enzyme engineering, discusses existing challenges, and looks forward to future research directions. Overall, this review aims to provide references and new ideas for research in related fields.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Broad-Spectrum Aqueous Esterification Using the Adenylation Domain of a Carboxylic Acid Reductase Coupled With ATP Regeneration","authors":"Kanokkan Sriwaiyaphram,&nbsp;Surawit Visitsatthawong,&nbsp;Nidar Treesukkasem,&nbsp;Pimchai Chaiyen,&nbsp;Thanyaporn Wongnate","doi":"10.1002/biot.70128","DOIUrl":"10.1002/biot.70128","url":null,"abstract":"<div>\u0000 \u0000 <p>Biocatalytic esterification in water is a green alternative to chemical synthesis but often faces challenges such as low enzyme efficiency, poor substrate solubility, and expensive cofactors. Here, we present a streamlined aqueous esterification system utilizing the adenylation domain of carboxylic acid reductase (A-domain_CAR), a minimal catalyst that efficiently activates carboxylic acids. A-domain_CAR exhibited superior catalytic performance over full-length CARs, achieving up to 96% yield of methyl cinnamate under optimized aqueous conditions. To improve cost-efficiency and scalability, the system was coupled with a Class III polyphosphate kinase 2 (Class III PPK2) from <i>Deinococcus proteolyticus</i> for in situ ATP regeneration using AMP and polyphosphate. This two-enzyme platform enabled high-yield esterification across a broad range of cinnamic and benzoic acid derivatives and various alcohols. Incorporating micellar media further enhanced the conversion of poorly soluble aromatic alcohols such as benzyl and phenethyl alcohol. Preparative-scale esterification of methyl caffeate, a bioactive antioxidant ester, was successfully demonstrated with a 66% yield in a 500 mL aqueous reaction. This study highlights A-domain_CAR as a modular, efficient, and scalable biocatalyst, advancing sustainable ester synthesis for applications in pharmaceuticals, fine chemicals, and bio-based materials.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211084","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Intracellular Antibody Levels Predict Secretion Efficiency in HEK293T Cells Expressing a Chimeric Anti-CD99 Antibody from a Single-Expression Plasmid","authors":"Manatchanok Chinakarapong,&nbsp;Kamonporn Kotemul,&nbsp;Sukrueta Hanprathet,&nbsp;Myint Myat Thu,&nbsp;Supansa Pata,&nbsp;Witida Laopajon,&nbsp;Watchara Kasinrerk,&nbsp;Nuchjira Takheaw","doi":"10.1002/biot.70129","DOIUrl":"10.1002/biot.70129","url":null,"abstract":"<div>\u0000 \u0000 <p>Recombinant antibody production in mammalian cells is essential for therapeutic, diagnostic, and research applications. Single-expression plasmid systems that co-express both heavy and light chains under balanced promoters offer advantages in improving expression consistency. However, the relationship between intracellular antibody expression levels and secretion efficiency following clonal selection remains unclear. In this study, a single-expression plasmid encoding a chimeric anti-CD99 antibody (ChAbMT99/1) was constructed and used to establish stably expressing HEK293T mini-pool populations through zeocin selection. Intracellular ChAbMT99/1 expression levels in five selected mini-pools were assessed using intracellular immunofluorescence staining and flow cytometry. Antibody secretion into the culture supernatants was quantified by indirect enzyme-linked immunosorbent assay (ELISA) and further validated using indirect immunofluorescence assay and flow cytometry with native CD99-expressing cells. The results revealed a strong positive correlation between intracellular antibody expression and secretion levels. Mini-pools with higher intracellular fluorescence intensities produced greater antibody yields, indicating that intracellular antibody expression levels, within the context of single-plasmid expression systems, can reliably predict secretion efficiency. This finding facilitates the efficient selection of high-producing mini-pools for large-scale recombinant antibody production.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Dynamics Simulation-Assisted Tuning of Key Sites to Alter the Substrate Specificity of the Aldo-Keto Reductase KmAKR","authors":"Shen-Yuan Xu,&nbsp;Yu-Feng Chen,&nbsp;Lei Cui,&nbsp;Zhen-Zhen Wu,&nbsp;Ren-Chao Zheng,&nbsp;Yu-Guo Zheng","doi":"10.1002/biot.70130","DOIUrl":"10.1002/biot.70130","url":null,"abstract":"<div>\u0000 \u0000 <p>Aldo-keto reductase (AKR) is an important biocatalyst for the synthesis of chiral alcohols; however, its inability to catalyze bulky substrates severely limits its industrial applications. Previously, the T23V/Q213A mutant of AKR from <i>Kluyveromyces marxianus</i> (<i>Km</i>AKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer-assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high-throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate-binding pocket, thereby influencing the substrate specificity of <i>Km</i>AKR.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211103","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Issue Information: Biotechnology Journal 10/2025","authors":"","doi":"10.1002/biot.70137","DOIUrl":"https://doi.org/10.1002/biot.70137","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70137","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145224078","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":"Developments in the Production of Recombinant Monoclonal Antibodies","authors":"Debajyoti Saha,&nbsp;Prabir Kumar Das,&nbsp;Venkata Dasu Veeranki","doi":"10.1002/biot.70127","DOIUrl":"10.1002/biot.70127","url":null,"abstract":"<div>\u0000 \u0000 <p>Over the last three decades, recombinant monoclonal antibodies (mAbs) have emerged as highly promising therapeutics in the treatment and cure of chronic diseases, such as cancer, cardiovascular disease, autoimmune diseases, sexually transmitted diseases, neurological disorders, ophthalmologic disorders, viral neutralization, research reagents, and diagnostic tools. This has led to a rapid increase in the mAb market globally. Increased market demands have led researchers to develop hosts like mammalian cells, bacteria, fungi, yeast, insect cell lines, transgenic plants, and transgenic animals for increased mAb production. To surpass different challenges related to activity and production, different upstream strategies like antibody engineering, host cell engineering, media formulation, and bioreactor parameter optimization are being developed. The development of simple and low-cost downstream techniques is also one of the most important steps in antibody production for making an economically feasible process and product. The present review aims to briefly discuss the history of mAb production and current strategies used for the development and production of recombinant mAbs on a larger scale.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145211106","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modifying Methylene-Tetrahydrofolate Reductase to Disrupt Electron Bifurcation in Clostridium autoethanogenum.","authors":"Lucas W Mendelson, Alexander P Mueller, Jeremy Vasquez, Steven D Brown, R Adam Thompson","doi":"10.1002/biot.70133","DOIUrl":"https://doi.org/10.1002/biot.70133","url":null,"abstract":"<p><p>Methylene-tetrahydrofolate reductase (MTHFR) is an important enzyme for acetogenic carbon fixation, but the redox mechanism driving this reaction is not clearly understood. Previous enzymology work and energetic accounting on species such as Clostridium autoethanogenum has led to confounding results when placed in the context of in vivo experiments. In this work, we create multiple C. autoethanogenum strains harboring alternative MTHFR enzyme complexes as well as genome-scale metabolic models to better understand how these organisms conserve energy on gas substrates. The inclusion of a Type-III MTHFR unexpectedly allows for higher growth than expected and suggests the possibility of an additional redox balancing cycle employed during autotrophic growth.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":"e70133"},"PeriodicalIF":3.1,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145237575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing Lentiviral Vector Production: Insights Into PiggyBac Transposase and Concatemeric Array Strategies.","authors":"Jona Röscheise, Maximilian Klimpel, Janina Hoffman, Vathsalya Pabbathi, Herbert Dersch, Parameswari Govindarajan, Holger Laux, Kerstin Otte","doi":"10.1002/biot.70135","DOIUrl":"10.1002/biot.70135","url":null,"abstract":"<p><p>Lentiviral vectors (LVVs) are essential tools in gene and cell therapy due to their ability to transduce both dividing and non-dividing cells. Conventional production by transient plasmid co-transfection is variable, costly, and difficult to scale, prompting development of stable producer cell lines. Historically, the GPRTG cell line has been generated using concatemeric-array integration, which requires high DNA input, complex workflows, and can cause genetic instability. To address these limitations, we evaluated a transposase-mediated integration strategy. Compared with the concatemeric-array method, transposase-based integration enabled faster recovery after selection with only a mild viability crisis and required substantially less DNA. This approach generated highly diverse and heterogeneous producer pools, providing a strong basis for subsequent clonal selection. During LVV production, both methods maintained comparable cell growth stability. However, concatemeric-derived pools exhibited greater variability in recovery kinetics, viable cell density, and LVV titers, despite achieving the highest maximum titers overall. In contrast, transposase-mediated pools showed more consistent performance, supporting their reliability for large-scale applications. In summary, transposase-based integration offers a robust and scalable alternative to concatemeric-array methods for generating stable LVV producer cell lines, with significant potential to streamline LVV manufacturing for gene therapy.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":"e70135"},"PeriodicalIF":3.1,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12501403/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145237567","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":"Characterization and Biolubricant Performance of Marinobacter alanticus Lipid Extract.","authors":"Eric O McGhee, Rebecca L Mickol, Lindsay Van House, Mark Romanczyk, Thomas Loegel, Kathryn J Wahl, Matthew D Yates","doi":"10.1002/biot.70134","DOIUrl":"10.1002/biot.70134","url":null,"abstract":"<p><p>Renewable and sustainable materials to replace petroleum-based lubricants are increasingly sought in tribology and energy applications. In this study, Marinobacter atlanticus cultures were harvested and cells extracted for intracellular lipids for subsequent combined chemical and tribological evaluation. Mass spectrometry revealed that the extract was dominated by the wax ester palmityl palmitoleate, confirming a high-molecular-weight ester profile favorable for boundary lubrication. Differential scanning calorimetry and thermogravimetric analysis indicated a glass transition at 0°C, a melting endotherm at 40°C, and an onset of thermal degradation near 220°C, providing a workable thermal window for moderate-temperature applications. Pin-on-disk tribometry of the neat extract yielded an average friction coefficient of <math> <semantics> <mrow><mover><mi>μ</mi> <mo>¯</mo></mover> <mo>=</mo> <mn>0.117</mn> <mspace></mspace> <mrow><mo>(</mo> <mrow><mo>±</mo> <mn>0.02</mn></mrow> <mo>)</mo></mrow> </mrow> <annotation>$bar{mu } = 0.117 ( { pm 0.02} )$</annotation></semantics> </math> and an average specific wear rate of <math> <semantics> <mrow><mover><mi>K</mi> <mo>¯</mo></mover> <mo>=</mo> <mn>1.1</mn> <mo>×</mo> <msup><mn>10</mn> <mrow><mo>-</mo> <mn>8</mn></mrow> </msup> <mrow><mo>(</mo> <mrow><mo>±</mo> <mn>2.9</mn> <mo>×</mo> <msup><mn>10</mn> <mrow><mo>-</mo> <mn>9</mn></mrow> </msup> </mrow> <mo>)</mo></mrow> <mi>m</mi> <msup><mi>m</mi> <mn>2</mn></msup> <msup><mi>N</mi> <mrow><mo>-</mo> <mn>1</mn></mrow> </msup> </mrow> <annotation>$bar{K} = 1.1 times {{10}^{ - 8}}( { pm 2.9 times {{{10}}^{ - 9}}} ){mathrm{m}}{{{mathrm{m}}}^2}{{N}^{ - 1}}$</annotation></semantics> </math> , values that compare favorably with conventional plant oils. Additionally, the bacterial lipid was blended into both poly-α-olefin (PAO) and polyol ester (POE) base stocks at several additive weight percentages. Low-level additions (<5 wt.%) further reduced friction relative to the neat bacterial extract and wear rate relative to the neat PAO, while the bacterial extract did not solubilize well into the POE. Collectively, the data demonstrate that lipids from M. atlanticus constitute a promising renewable drop-in additive or standalone biolubricant for industrial formulations, advancing the transition toward sustainable tribological materials.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":"e70134"},"PeriodicalIF":3.1,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12501400/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145237593","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":"Low Concentration Ionic Liquid-Assisted Efficient Enzymatic Degradation of Xanthan.","authors":"Chunshu Chen, Fan Yang, Le Zhong, Jinyun Gu, Weiming Liu, Wenbo Li, Ruiyu Shen, Zhimin Yu","doi":"10.1002/biot.70136","DOIUrl":"https://doi.org/10.1002/biot.70136","url":null,"abstract":"<p><p>One of the main obstacles to preparing oligoxanthan through the enzymatic degradation of xanthan gum (XG) is its dense structure. To achieve the large-scale production of oligoxanthan, it is necessary to perturbate the conformation of XG and increase enzyme accessibility. This research developed an efficient system for the enzymatic degradation of XG assisted by low concentrations of ionic liquids (ILs). Structural characterization results indicated that a low concentration of [EMIM]DEP (1%, v/v) not only loosens the structure of XG but also alters the secondary structure of enzyme, thereby dramatically increasing its hydrolytic activity and eventually enhancing the production of oligoxanthan. The results were further elucidated by molecular dynamics (MD) simulation. Under the hydrolysis conditions of 1.5 mg/mL Aspergillus niger cellulase (AnCel), 0.2 mg/mL XG, and 1% (v/v) [EMIM]DEP at 40°C and pH 5.0 for 20 min, XG can be hydrolyzed into a mixture with a glucose equivalent of 32.25%. The degree of polymerization (DP) of XG hydrolysates was 3-10. Moreover, the xanthan hydrolysates obtained using this system exhibit excellent antioxidant activity. This study proposed an effective method for the preparation of functional oligosaccharides, which is of significant importance for the valorization of XG.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":"e70136"},"PeriodicalIF":3.1,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145256912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}