Biotechnology Journal最新文献

{"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}

{"title":"An Experimental and Modeling Approach to Study Tangential Flow Filtration Performance for mRNA Drug Substance Purification","authors":"Ehsan Nourafkan,&nbsp;Charlotte Kenyon,&nbsp;Adithya Nair,&nbsp;Kate A. Loveday,&nbsp;Emma N. Welbourne,&nbsp;Min Tao,&nbsp;Mahdi Ahmed,&nbsp;Joseph Middleton,&nbsp;Mark J. Dickman,&nbsp;Solomon F. Brown,&nbsp;Mabrouka Maamra,&nbsp;Joan Cordiner,&nbsp;Zoltán Kis","doi":"10.1002/biot.202400473","DOIUrl":"10.1002/biot.202400473","url":null,"abstract":"<p>Following the recent COVID-19 pandemic, mRNA manufacturing processes are being actively developed and optimized to produce the next generation of mRNA vaccines and therapeutics. Herein, the performance of the tangential flow filtration (TFF) was evaluated for high-recovery, and high-purity separation of mRNA from unreacted nucleoside triphosphates (NTPs) from the in vitro transcription (IVT) reaction mixture. For the first time, the fouling model was successfully validated with TFF experimental data to describe the adsorption of mRNA on filtration membrane. The fouling model enables monitoring of the mRNA purification processes, designing an appropriate strategy for filter clean-up, replacing the column at the right time and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ∼19 g/m², &lt;2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach also enables the purification of multiple mRNA drug substance sequences for the treatment of a wide range of different diseases.</p>","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.202400473","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602132","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 Regioselectivity of P450 BM3 Enables the Biosynthesis of Murideoxycholic Acid by 6β-Hydroxylation of Lithocholic Acid","authors":"Fangzhi Deng,&nbsp;Zhenru Zhou,&nbsp;Zhen Du,&nbsp;Mohamed Mohany,&nbsp;Qunyue Wu,&nbsp;Weiyang Liang,&nbsp;Lei Zhang,&nbsp;Shan Li","doi":"10.1002/biot.202400518","DOIUrl":"10.1002/biot.202400518","url":null,"abstract":"<div>\u0000 \u0000 <p>Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from <i>Bacillus megaterium</i>, employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.</p>\u0000 </div>","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":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602143","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":"Genome-Scale Modeling of CHO Cells Unravel the Critical Role of Asparagine in Cell Culture Feed Media","authors":"Kuin Tian Pang,&nbsp;Yi Fan Hong,&nbsp;Fumi Shozui,&nbsp;Shunpei Furomitsu,&nbsp;Matthew Myint,&nbsp;Ying Swan Ho,&nbsp;Yaron R. Silberberg,&nbsp;Ian Walsh,&nbsp;Meiyappan Lakshmanan","doi":"10.1002/biot.202400072","DOIUrl":"10.1002/biot.202400072","url":null,"abstract":"<div>\u0000 \u0000 <p>Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized a CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conducted in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.</p>\u0000 </div>","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":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602153","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}