Biotechnology Journal最新文献

{"title":"Multilevel Metabolic Engineering of Cupriavidus necator for CO2-Derived Single-Cell Protein Production","authors":"Menghan Chang,&nbsp;Zhiguo Huang,&nbsp;Shiqi Liu,&nbsp;Beini Yang,&nbsp;Ranran Wu,&nbsp;Lijuan Ma,&nbsp;Lei Chen,&nbsp;Zhiguang Zhu","doi":"10.1002/biot.70320","DOIUrl":"10.1002/biot.70320","url":null,"abstract":"<div>\u0000 \u0000 <p>Enhancing nitrogen assimilation and coordinating carbon-energy metabolism in the autotrophic chassis <i>Cupriavidus necator</i> remain key challenges for maximizing single-cell protein (SCP) production from CO₂. In this study, multilevel engineering was performed on <i>C. necator</i>. First, <i>glnA</i> was overexpressed to enhance nitrogen assimilation, which increased crude protein content to 72.75% in the best-performing strain, GS13. Preliminary physiological characterization suggested that this increase correlated with enhanced heme level. Second, <i>glcR</i> was disrupted to alter carbon allocation toward amino acid precursor metabolism, and <i>epr-1</i> was deleted to inhibit extracellular protein degradation during the secretion process. The double-knockout strain Δ<i>epr-1</i>Δ<i>glcR</i> achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD₆₀₀ increased to 13.12 while crude protein content decreased to 68.05%, indicating a non-additive interaction between the engineered modules. Overall, our study provided improved strains and a modular framework for future SCP optimization and mechanistic studies of carbon-nitrogen coordination in <i>C. necator</i>.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178182","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":"Recent Advances in Engineering Ammonia Lyase for Biosynthesis of Aromatic Noncanonical Amino Acids","authors":"Wen Zhang,&nbsp;Yu Sun,&nbsp;Zhen-Yu Liao,&nbsp;Bin Xue,&nbsp;Shu-Yun Ju,&nbsp;Yan-Jun Wan,&nbsp;Ya-Jun Wang","doi":"10.1002/biot.70321","DOIUrl":"10.1002/biot.70321","url":null,"abstract":"<div>\u0000 \u0000 <p>Aromatic noncanonical amino acids (aromatic ncAAs) are valuable building blocks for pharmaceuticals, agrochemicals, food additives, and specialty chemicals, yet their sustainable and stereoselective synthesis remains challenging. Ammonia-lyases (ALs) have emerged as powerful biocatalysts for the one-step asymmetric hydroamination of arylacrylic acids, and recent advances in protein engineering have substantially enhanced their catalytic performance including efficiency, selectivity, promiscuity, and productivity. This review integrates catalytic mechanisms with the deployment of engineering strategies to illustrate how aromatic amino acid ammonia-lyases (AALs) have evolved into versatile biocatalysts for aromatic ncAAs synthesis. Particular attention is devoted to the engineering of AALs (including phenylalanine, tyrosine, histidine ammonia lyase) and (methyl)aspartate ammonia lyases, highlighting progress in substrate scope expansion, catalytic promiscuity, selectivity modulation, and hybrid catalytic systems. Current limitations and emerging opportunities are further assessed to delineate future directions for next-generation ALs engineering. The engineering strategies and current states of ALs summarized herein provide a conceptual basis for the efficient development of ALs for sustainable aromatic ncAAs biomanufacturing.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178175","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":"Efficient and High-Resolution Separation of Partial, Empty, and Full AAV Capsids by Optimized Iodixanol Density Gradient Ultracentrifugation","authors":"Shan Fang,&nbsp;Jun Wu,&nbsp;Yanfang Shen,&nbsp;Dandan Qi,&nbsp;Zhuoyu Ni,&nbsp;Fenghua Wang","doi":"10.1002/biot.70319","DOIUrl":"10.1002/biot.70319","url":null,"abstract":"<div>\u0000 \u0000 <p>Recombinant adeno-associated virus (rAAV) vectors are essential for gene therapy, but their production generates empty and partial capsids as product-related impurities that can compromise efficacy and safety. While empty capsids have been partially characterized, the isolation and study of partial capsids remain hindered by a lack of robust separation methods. Here, we present an optimized iodixanol density gradient ultracentrifugation strategy enabling high-resolution separation and enrichment of empty, partial, and full AAV capsids. Key innovations include expanding the 40% iodixanol layer to create a broader intermediate-density zone and implementing controlled bottom-puncture fractionation. Applied to a rAAV2 preparation, this method enriched partial capsids from 18.9% in the starting material to approximately 94.9% in the target fraction (determined by AUC integration under the equal response assumption). For a rAAV2 variant with a low baseline of partial capsids (1.3%), over 20-fold enrichment was achieved, reaching 30.1% purity as determined by AUC integration under the equal response assumption. This approach provides partial-capsid-enriched fractions, enabling detailed characterization of partial capsids, supporting critical quality attribute assessment, and facilitating the development of safer, more effective rAAV-based therapeutics.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178200","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":"Role of Calpain in Autolysis of Sea Cucumber Apostichopus japonicus","authors":"Siyu Yao,&nbsp;Siyuan Ma,&nbsp;Guanhua Yan,&nbsp;Ting Zhao,&nbsp;Yiying Nian,&nbsp;Ling Zhang,&nbsp;Liming Sun","doi":"10.1002/biot.70286","DOIUrl":"10.1002/biot.70286","url":null,"abstract":"<div>\u0000 \u0000 <p>Calpain is a family of cysteine proteases whose role in sea cucumber autolysis was previously unreported. In this study, specific inhibition of calpain notably alleviated autolysis in the respiratory tree and intestine, significantly reduced body wall hardness loss. During an 8 h autolysis period, calpain activity in the body wall continuously declined, whereas activity in the respiratory tree and intestine peaked at 2 h. The contents of calpain 5 &amp; 9 were highest in the respiratory tree and remained relatively stable, whereas these two isoforms showed distinct temporal patterns in the intestine and body wall. Immunohistochemistry revealed high calpain expression in the muscle layers and at the mucosal fold tips of the intestine and respiratory tree, but sparse distribution in the body wall connective tissue. After 4 h of autolysis, disintegration of the intestinal serosal layer, disorganization of the respiratory tree cellular structure, and destruction of the epidermis and endodermis of the body wall were observed. Co-immunoprecipitation coupled with mass spectrometry identified 40 calpain-interacting proteins, and fluorescence colocalization confirmed direct interaction with tubulin. This first report establishes calpain involvement in sea cucumber autolysis, clarifying its dynamic activity, tissue distribution, and interacting proteins, and suggests calpain may act by degrading tubulin.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178213","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":"An Integrated Transient Expression and UPLC-PDA Approach for Functional Genomics and Metabolic Analysis of Cannabis sativa","authors":"Sarah Pullano,&nbsp;Aida Shahabi,&nbsp;Parmis Shariat Torbaghani,&nbsp;Ivan Monsalvo,&nbsp;Madison Sandler,&nbsp;Adam Sumner,&nbsp;Alex Harris,&nbsp;David Haak,&nbsp;Bastiaan Bargmann,&nbsp;Nik Kovinich","doi":"10.1002/biot.70316","DOIUrl":"10.1002/biot.70316","url":null,"abstract":"<p><i>Cannabis sativa</i> (<i>Cannabis</i>) is a high-value medicinal crop whose functional genomics and metabolic engineering remain constrained by limited genetic tools. The species is regarded as recalcitrant to stable transformation, and existing transient expression systems exhibit low efficiency or are restricted to hemp cultivars. Here, we report two <i>Agrobacterium</i>-mediated agroinfiltration protocols enabling rapid, robust, and reproducible transient gene expression in drug-type, high-THC <i>Cannabis</i> tissues. Using the <i>RUBY</i> betalain reporter system, we demonstrate transformation efficiencies of up to 100% in mature leaf disks and 81%–100% in whole seedlings. By independently comparing <i>Agrobacterium</i> strains and vector configurations, we demonstrate that transformation efficiency and transgene expression are influenced by distinct experimental variables, with EHA105 providing the highest transformation efficiency and <i>p35S</i>-based vectors producing the strongest reporter expression. Complementing these genetic tools, we developed a UPLC-PDA method enabling simultaneous detection and quantification of cannabinoids and cannflavins. While not intended to replace mass spectrometry-based analyses, the method provides an accessible and cost-effective platform for comparative metabolite profiling during functional genomics and metabolic engineering studies. Together, these advances establish an integrated workflow linking high-efficiency transient gene expression with streamlined biochemical analysis, providing a practical foundation for gene function studies, pathway interrogation, and biotechnology-driven trait engineering in <i>Cannabis</i>.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70316","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178205","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":"Sticking to the Heart: Adhesive Hydrogels for Cardiac Regeneration","authors":"Niayesh Najafi,&nbsp;Kevin Babakhan Vartanian,&nbsp;Tony Eskandar,&nbsp;Edgmin Rostomian,&nbsp;Finosh G. Thankam","doi":"10.1002/biot.70315","DOIUrl":"10.1002/biot.70315","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Purpose</h3>\u0000 \u0000 <p>Adhesive hydrogels (AHs) are promising biomaterials for cardiac tissue engineering due to their injectability, ability to adhere to wet tissue, and structural similarity to the native extracellular matrix (ECM). This review evaluates the perspectives, opportunities, and challenges associated with AHs in cardiac tissue repair and functional recovery. Adhesion is achieved through chemical mechanisms including Schiff base reactions, Michael addition, and enzymatic crosslinking, as well as physical interactions including hydrogen bonding, host–guest complexation, and catechol-based binding. These mechanisms support integration with the dynamic and mechanically active cardiac surface ensuring the localized and sustained delivery of cells/therapeutics.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Peer reviewed articles were identified through searches of PubMed and Embase using terms related to “adhesive hydrogels,” “bioadhesion,” and “cardiac tissue engineering.” Studies were included based on relevance to adhesion chemistry, mechanical integration, in vivo cardiac performance, and translational outcomes.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Recent reports demonstrated improvements in ventricular remodeling, neovascularization, and scar size reduction, while early-stage clinical trials report safety and potential functional benefits of AH-based cardiac applications.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>Continued progress in material design, biocompatibility, and clinical delivery approaches is essential for translating adhesive hydrogels into routine therapies for heart management.</p>\u0000 </section>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149101523","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":"In Situ Imaging for Monitoring Biomass Content and Pellet Size in Submerged Filamentous Stirred-Tank Cultivation","authors":"Lukas Hartmann,&nbsp;Tarek Wieland,&nbsp;Linus Witucki,&nbsp;Mike Barth,&nbsp;Dirk Holtmann","doi":"10.1002/biot.70314","DOIUrl":"https://doi.org/10.1002/biot.70314","url":null,"abstract":"<p>Filamentous microorganisms play a central role in industrial biotechnology, where pellet morphology affects mass transfer, rheology, and process performance. However, time-resolved monitoring of filamentous biomass and pellet size dynamics in stirred-tank reactors remains challenging due to offline sampling limitations. We present a noninvasive in situ imaging approach to observe biomass-related light scattering and projected pellet size. A custom-built camera and ring-light setup with MATLAB-based image acquisition and processing was used to quantify image luminosity as a proxy for biomass content and to determine projected object area (POA) and the number of detected objects per image above a POA threshold of 0.5 mm². To characterize systematic biases, polyoxymethylene spheres of 1–4 mm diameter served as a model system. Luminosity scaled with object concentration, whereas object detection and apparent size estimation were increasingly affected by object size and concentration. In a bench-scale cultivation of malic acid-producing <i>Aspergillus oryzae</i>, image luminosity followed biomass content, while in situ POA analysis revealed increasing projected pellet size coinciding with Zn²⁺ limitation. These observations differed from ex situ sampling, indicating that image-based in situ monitoring provides time-resolved information on biomass formation and pellet size dynamics that may not be captured by offline sampling alone.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70314","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149068527","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 rAAV Production in HEK293 Cells by Promoting Helper Plasmid Nuclear Import Under HDAC6 Inhibition","authors":"He Ren,&nbsp;Jianqi Nie,&nbsp;Zichuan Song,&nbsp;Wanting Mo,&nbsp;Yankun Yang,&nbsp;Zhonghu Bai","doi":"10.1002/biot.70313","DOIUrl":"https://doi.org/10.1002/biot.70313","url":null,"abstract":"<div>\u0000 \u0000 <p>Recombinant adeno-associated virus (rAAV) vectors have emerged as leading platforms for gene therapy owing to their safety, long-term transgene expression, and broad tissue tropism. However, rAAV production in HEK293 cells via triple-plasmid transfection remains limited by low yield, partly due to inefficient nuclear import of plasmids. In this study, we enhanced rAAV production by promoting plasmid nuclear delivery. DNA-targeting sequence (DTS) insertion alone did not improve plasmid nuclear import or rAAV yield, whereas combining DTS insertion with tubacin, an HDAC6 inhibitor, significantly increased nuclear plasmid accumulation and rAAV productivity. Among the three plasmids, inserting the 5NF sequence into pHelper produced the greatest effect. Under tubacin treatment, pHelper-5NF increased rAAV5 yield from 1.08 × 10¹¹ to 2.44 × 10¹¹ vg/mL (2.26-fold), without altering the full capsid percentage. This improvement was associated with increased Rep/Cap expression and viral DNA replication. Moreover, the pHelper-5NF enhanced rAAV production across multiple capsid serotypes, including rAAV1, rAAV2, rAAV6, rAAV8, rAAV9, and rAAVrh10. Unexpectedly, insertion of 5NF into two or three plasmids did not further increase rAAV yield via triple-plasmid transfection. These results identify pHelper-5NF combined with HDAC6 inhibition as an effective strategy to enhance triple-plasmid transfection-based rAAV manufacturing.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149068528","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 10/2026","authors":"","doi":"10.1002/biot.70318","DOIUrl":"https://doi.org/10.1002/biot.70318","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 10","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70318","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149068524","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":"Mammalian Expression and Mass Spectrometric Characterization of Highly Active Human Arylsulfatase A","authors":"T. Pal,&nbsp;M. Dabarera,&nbsp;A. Dutta,&nbsp;G. Smolenski,&nbsp;B. D. Dickson,&nbsp;A. Williamson,&nbsp;W. Kelton","doi":"10.1002/biot.70312","DOIUrl":"10.1002/biot.70312","url":null,"abstract":"<p>The lysosomal enzyme Arylsulfatase A (ARSA) plays a critical role in hydrolyzing sulfatides from within cells. While gene therapies for ARSA replacement have recently been approved, challenges remain in recombinantly producing large amounts of active ARSA, especially for research purposes where generating stable cell lines is cumbersome. A major bottleneck is the need to convert the active-site residue Cysteine at position 69 to formylglycine by a formylglycine-generating enzyme (FGE). Further, transporter mannose-6-phosphate receptors (M6PR) are required to mediate active transport of ARSA out of the cell. To address these issues, we have optimized a three-plasmid transient transfection approach that produces ARSA with active site conversion of over 70% as measured by mass spectrometry and is highly active in assays with a model substrate. We find the optimal ARSA:M6PR:FGE plasmid ratio to be 8:1:1, which balances active site conversion with total protein yield. This optimized protocol will assist in producing highly active ARSA for biochemical studies in research labs with access to standard cell culture facilities.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"21 9","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70312","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149035795","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}