Biotechnology Journal最新文献

{"title":"Issue Information: Biotechnology Journal 12/2024","authors":"","doi":"10.1002/biot.202470121","DOIUrl":"https://doi.org/10.1002/biot.202470121","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470121","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142868148","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":"A Competition-Based Strategy for the Isolation of an Anti-Idiotypic Blocking Module and Fine-Tuning for Conditional Activation of a Therapeutic Antibody","authors":"Jan Habermann,&nbsp;Dominic Happel,&nbsp;Adrian Bloch,&nbsp;Charles Shin,&nbsp;Harald Kolmar","doi":"10.1002/biot.202400432","DOIUrl":"10.1002/biot.202400432","url":null,"abstract":"<p>The masking of therapeutic antibodies by the installation of a blocking module that can be removed under certain physiological conditions, is becoming increasingly important to improve their safety and toxicity profile. To gain access to such masking units, we used chicken immunization in combination with yeast surface display and a competition-based FACS screening campaign to obtain anti-idiotypic single-chain Fv (scFv) fragments. This approach promotes the identification of functional masking units, since specificity and high affinity do not necessarily guarantee a paratope blocking effect. This strategy was used to isolate a scFv masking unit for the therapeutic antibody 6G11 (BI-1206), which is currently in clinical trials for the treatment of B-cell lymphoma to block the inhibitory Fcγ receptor IIB (CD32b). N-terminal fusion of the anti-idiotypic scFv to the 6G11 light chain successfully abolished binding to FcγRIIB in vitro. For conditional activation, a cleavable linker for the tumor-associated protease MMP-9 was implemented. To improve demasking efficiency, the affinity of the scFv mask was attenuated through rational design. The substitution of one key amino acid in the masking scFv reduced the affinity toward the 6G11 paratope by factor 10 but still mediated 9800-fold blocking of receptor binding. Proteolytic demasking allowed full recovery of therapeutic antibody function in vitro, supporting the concept of conditional antibody activation using this anti-idiotypic binding module.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629141/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798849","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":"Selective Recruitment of a Synthetic Histone Acetyltransferase Can Boost CHO Cell Productivity","authors":"Sienna P. Butterfield,&nbsp;Rebecca E. Sizer,&nbsp;Fay L. Saunders,&nbsp;Robert J. White","doi":"10.1002/biot.202400474","DOIUrl":"10.1002/biot.202400474","url":null,"abstract":"<p>Industrial production of biologics typically involves the integration of transgenes into host cell genomes, most often Chinese hamster ovary (CHO) cells. Epigenetic control of transgene expression is a major determinant of production titers. Although the cytomegalovirus (CMV) promoter has long been used to drive industrial transgene expression, we found that its associated histones are suboptimally acetylated in CHO cells, providing an opportunity to enhance productivity through epigenetic manipulation. Expression of monoclonal antibody mRNAs increased up to 12-fold when a CRISPR-dCas9 system delivered the catalytic domain of a histone acetyltransferase to the CMV promoter. This effect was far stronger than when promoter DNA was selectively demethylated using dCas9 fused to a 5-methylcytosine dioxygenase. Mechanistically, acetylation-mediated transcriptional activation involved heightened phosphorylation and activity of RNA polymerase II, enabling it to escape promoter-proximal pausing at the transgene. This approach almost doubled the titer and specific productivity of antibody-producing CHO cells, demonstrating the potential for biomanufacturing.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629143/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142798851","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":"Autophagy and Akt-Stimulated Cellular Proliferation Synergistically Improve Antibody Production in CHO Cells","authors":"Leran Mao,&nbsp;Sarah Michelle Sonbati,&nbsp;James W. Schneider,&nbsp;Anne S. Robinson","doi":"10.1002/biot.202400033","DOIUrl":"https://doi.org/10.1002/biot.202400033","url":null,"abstract":"<p>Over the past decade, engineered producer cell lines have led 10-fold increases in antibody yield, based on an improved understanding of the cellular machinery influencing cell health and protein production. With prospects for further production improvements, increased antibody production would enable a significant cost reduction for life-saving therapies. In this study, we strategized methods to increase cell viability and the resulting cell culture duration to improve production lifetimes. By overexpressing the cell surface adenosine A_2A receptor (A_2AR), the Akt pathway was activated, resulting in improved cellular proliferation. Alternatively, by inducing autophagy through temperature downshift, we were able to significantly enhance cellular-specific productivity, with up to a three-fold increase in total antibody production as well as three-fold higher cell-specific productivity. Interestingly, the expression levels of the autophagy pathway protein Beclin-1 appeared to correlate best with the total antibody production, of autophagy-related proteins examined. Thus, during cell clonal development Beclin-1 levels may serve as a marker to screen for conditions that optimize antibody titer.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400033","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763996","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 of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress","authors":"Karuppuchamy Selvaprakash,&nbsp;Christiana-Kondylo Sideri,&nbsp;Michael Henry,&nbsp;Esen Efeoglu,&nbsp;David Ryan,&nbsp;Paula Meleady","doi":"10.1002/biot.202400413","DOIUrl":"https://doi.org/10.1002/biot.202400413","url":null,"abstract":"<p>Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified &gt;4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of &gt;900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, <i>N</i>-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400413","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763999","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":"Engineering Saccharomyces boulardii for Probiotic Supplementation of l-Ergothioneine","authors":"Chaoqun Tang,&nbsp;Lu Zhang,&nbsp;Junyi Wang,&nbsp;Congjia Zou,&nbsp;Yalin Zhang,&nbsp;Jifeng Yuan","doi":"10.1002/biot.202400527","DOIUrl":"10.1002/biot.202400527","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Saccharomyces boulardii</i>, as a probiotic yeast, has shown great potential in regulating gut health and treating gastrointestinal diseases. Due to its unique antimicrobial and immune-regulating functions, it has become a significant subject of research in the field of probiotics. In this study, we aim to enhance the antioxidant properties of <i>S. boulardii</i> by producing <span>l</span>-ergothioneine (EGT). We first constructed a double knockout of <i>ura3</i> and <i>trp1</i> gene in <i>S. boulardii</i> to facilitate plasmid-based expressions. To further enable effective genome editing of <i>S. boulardii</i>, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of <i>S. boulardii</i>. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. By using PiggyBac transposon system, a great variety of EGT-producing strains was obtained, which is not possible for the conventional single target genome editing, and one best isolated top producer reached 17.50 mg/L EGT after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to <i>S. boulardii</i> for the first time for genetic engineering. The engineered probiotic yeast <i>S. boulardii</i> has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142674625","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":"Construction of a Cell Factory for the Targeted and Efficient Production of Phytosterol to Boldenone in Mycobacterium neoaurum","authors":"Bo Zhang,&nbsp;Sifang Zhu,&nbsp;Yi Zhu,&nbsp;Xin Sui,&nbsp;Junping Zhou,&nbsp;Zhiqiang Liu,&nbsp;Yuguo Zheng","doi":"10.1002/biot.202400489","DOIUrl":"10.1002/biot.202400489","url":null,"abstract":"<div>\u0000 \u0000 <p>Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ¹-dehydrogenase was improved through enzyme engineering, and their expression in the new strain of <i>Mycobacterium neoaurum</i> was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene <i>kasB</i> was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646071","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":"L-Asparaginase from Lachancea Thermotolerans: Effect of Lys99Ala on Enzyme Performance and in vitro Antileukemic Efficacy","authors":"Berin Yilmazer Aktar,&nbsp;Arzu Aysan,&nbsp;Ossi Turunen,&nbsp;Tamer Yağci,&nbsp;Hüseyin Avni Solğun,&nbsp;Barış Binay","doi":"10.1002/biot.202400507","DOIUrl":"10.1002/biot.202400507","url":null,"abstract":"<div>\u0000 \u0000 <p>L-asparaginases (EC 3.5.1.1) are amidohydrolase enzymes that predominantly catalyze conversion of L-asparagine to L-aspartic acid and ammonia. In addition, some exhibit secondary L-glutaminase activity. <i>Escherichia coli</i> and <i>Erwinia chrysanthemi</i> L-asparaginases are widely used in the pharmaceutical industry to produce therapeutically important compounds. In the therapeutic use of enzymes, bacterial L-asparaginases can trigger immune responses, leading to a high rate of adverse effects that diminish the effectiveness of the treatment. This situation has forced scientists to search for promising L-asparaginases from new sources. Yeast L-asparaginases could be useful in reducing toxicity and enhancing efficacy but they have been poorly studied to date. Here, we characterized the yeast <i>Lachancea thermotolerans</i> L-asparaginase (<i>Lt</i>ASNase) purified by affinity chromatography. It has a specific activity of 313.8 U/mg and a high k_cat value (312.4 s). We demonstrated through a semi-rational design that the mutations of Lys99 show varying effects on catalytic activity, with the Lys99Ala mutant increasing specific activity 3.3-fold. Furthermore, the in vitro antileukemic activity of the non-formulated form of Lys99Ala <i>Lt</i>ASNase was evaluated against SUP-B15 and REH cell lines. The results demonstrated that <i>Lt</i>ASNase exhibits significant antileukemic potential, comparable to commercial type II bacterial enzymes. The understanding of the mutant L-asparaginases examined in this study will significantly contribute to the development of new and more effective yeast-derived asparaginases.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646073","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":"Multifunctional PAMAM Dendrimers Carrying SAHA, 5-FU, and a Therapeutic Gene for Targeted Co-Delivery Toward Colorectal Cancer Cells","authors":"Bünyamin Bulkurcuoğlu,&nbsp;Mustafa Ulvi Gürbüz,&nbsp;Silvia Tyciakova,&nbsp;Kristina Pavlov,&nbsp;Nikoleta Mojzesova,&nbsp;Miroslava Matuskova,&nbsp;Metin Tülü,&nbsp;Şebnem Erçelen","doi":"10.1002/biot.202400362","DOIUrl":"10.1002/biot.202400362","url":null,"abstract":"<p>A promising approach to treat colorectal cancer (CRC) involves combining chemotherapy, epigenetics, and gene therapy to combat drug resistance. Multifunctional nanocarriers have emerged as a valuable tool for targeted CRC therapy. By delivering multiple treatments directly to cancer cells, these nanocarriers offer the potential for improved outcomes and reduced side effects. PAMAM-based dendrimers were functionalized with a unique combination of folic acid, 5-FU, SAHA, and plasmid DNA pCIneoGFP for targeted delivery to CRC cells. Biophysical characterizations of therapeutic loaded dendrimers and their complexes with pCIneoGFP were performed by: dynamic light scattering, fluorescence spectroscopy, and gel electrophoresis. Further, cellular analyses of dendriplexes demonstrated high transfection efficiency and anticancer activity on HCT 116 and HT-29 cell lines. We have successfully developed a multifunctional nanocarrier platform based on PAMAM dendrimers, offering a promising tool for targeted combination therapy of CRC.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400362","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646075","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":"Issue Information: Biotechnology Journal 11/2024","authors":"","doi":"10.1002/biot.202470111","DOIUrl":"https://doi.org/10.1002/biot.202470111","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 11","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142664610","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}