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":"Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.","authors":"Pu Yuan, Muhammad Usman, Wenshan Liu, Ashna Adhikari, Chunquan Zhang, Victor Njiti, Ye Xia","doi":"10.1002/biot.202400457","DOIUrl":"https://doi.org/10.1002/biot.202400457","url":null,"abstract":"<p><p>Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":"e202400457"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845602","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":"Considerations Regarding High Oil Density Bioreactor-Scale Fermentations of Yarrowia lipolytica Using CFD Modeling and Experimental Validation.","authors":"Sarah M Coleman, Richard J Marx, Morgan K Martinez, Ashli J Silvera, Junwon Park, Esha Ramanan, Geena Kaown, Seongkyu Yoon, Dongming Xie, Hal S Alper","doi":"10.1002/biot.202400506","DOIUrl":"https://doi.org/10.1002/biot.202400506","url":null,"abstract":"<p><p>Hydrophobic feedstocks such as waste cooking oil have recently been considered for microbial biotransformation due to their abundance, low cost, and unique advantage for lipid-derived fermentation products. Most fermentations with hydrophobic substrates are conducted at the tube or flask scale (less than 1 L total volume) or with the hydrophobic substrate comprising a small fraction of the media. Low substrate concentrations require additional feeding. Alternatively, high concentrations do not require significant dilution of the oil feedstock, which reduce volumetric requirements for larger scale fermentations. However, high-oil-density fermentations complicate efficient mixing and mass transfer challenges which are exacerbated at larger scales. To address this, computational fluid dynamics (CFD) models were explored to simulate three-phase (hydrophobic, hydrophilic, and gaseous) bench (3 L) and pilot scale (4000 L) bioreactors, highlighting challenges and potential considerations. Bioreactor fermentations of Yarrowia lipolytica strain L36DGA1 with substrate loadings of 5%, 10%, 20%, 30%, 40%, and 50% (v/v) waste cooking oil were also conducted, representing one of the highest concentrations in the reported literature. This work supports future research into and implementation of high-oil-density fermentations at the bench and pilot bioreactor scale.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":"e202400506"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142826870","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":"Creating a System of Dual Regulation of Translation and Transcription to Enhance the Production of Recombinant Protein.","authors":"Xin Li, Peng-Wei Shi, Fei Du, Zi-Xu Zhang, Zi-Jia Li, Na Wu, Guang Yang, Wang Ma, Xiao-Man Sun","doi":"10.1002/biot.202400679","DOIUrl":"https://doi.org/10.1002/biot.202400679","url":null,"abstract":"<p><p>When constructing cell factories, it is crucial to reallocate intracellular resources towards the synthesis of target compounds. However, imbalanced resource allocation can lead to a tradeoff between cell growth and production, reducing overall efficiency. Reliable gene expression regulation tools are needed to coordinate cell growth and production effectively. The orthogonal translation system, developed based on genetic code expansion (GCE), incorporates non-canonical amino acids (ncAAs) into proteins by assigning them to expanded codons, which enables the control of target protein expression at the translational level in an ncAA-dependent manner. However, the stringency of this regulatory tool remains inadequate. This study achieved strict translational-level control of the orthogonal translation system by addressing the abnormal leakage caused by the arabinose-inducible promoter. Further validation was conducted on the relationship between ncAA concentration and expression level, as well as the host's adaptability to the system. Subsequently, the system's applicability across multiple Escherichia coli hosts was verified by examining the roles of RF1 (peptide chain release factor 1) and endogenous TAG codons. By combining this strategy with inducible promoters, dual-level regulation of target gene expression at both transcriptional and translational levels was achieved and the dynamic range was further increased to over 20-fold. When using ncAA to control the expression of T7 RNA polymerase (T7 RNAP), the leakage expression was reduced by 82.7%, mitigating the low production efficiency caused by extensive leakage in the T7 system. As proof of concept, the strategy enhanced the production of alcohol dehydrogenase (ADH) by 9.82-fold, demonstrating its excellent capability in controlling gene expression in developing cell factories.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":"e202400679"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142826876","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":"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, Dominic Happel, Adrian Bloch, Charles Shin, Harald Kolmar","doi":"10.1002/biot.202400432","DOIUrl":"10.1002/biot.202400432","url":null,"abstract":"<p><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":"e202400432"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","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, Rebecca E Sizer, Fay L Saunders, Robert J White","doi":"10.1002/biot.202400474","DOIUrl":"10.1002/biot.202400474","url":null,"abstract":"<p><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":"e202400474"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","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":"Visualize PIM-1 Protein Function and Its Interaction With PI3K/Akt/mTOR Pathway Regulated by Its Active Sites Through FRET Biosensors.","authors":"Na Li, Youyi Zhao, Danbo Wang, Shuai Shao, Zhengyao Zhang, Bo Liu","doi":"10.1002/biot.202400443","DOIUrl":"https://doi.org/10.1002/biot.202400443","url":null,"abstract":"<p><p>Pro-viral Insertion site for the Moloney Murine Leukemia virus 1 (PIM-1) is widely involved in various biological processes and diseases, which is based on its structure and functional sites. However, the relationship between active sites and function of PIM-1 kinase remains unclear due to the lack of effective study approaches in live cells. Herein, to visualize the effect of different active sites in PIM-1 protein on its function activity and relation with PI3K/Akt/mTOR pathway, three mutant probes of EPHY which was developed previously based on fluorescence resonance energy transfer (FRET) technology to detect PIM-1 kinase activity in living cells were further constructed and transfected into cells followed by treating with PIM-1 inhibitors, ATP and PI3K inhibitor, respectively. The results showed that Lys67 is related to substrate binding and catalytic activity of PIM-1 kinase, thereby directly regulating PI3K/Akt/mTOR signaling pathway. Pro81/Asn82 are primarily participated in PIM-1 binding to ATP, thus also involving in the modulation on PI3K/Akt/mTOR signaling pathway, but play less role in the interaction between PIM-1 protein and its substrate. Asp167 has few effects on both the catalytic function activity of PIM-1 and PI3K/AKT/mTOR pathway, even though the binding ability of PIM-1 protein to its substrate is dramatically inhibited by D167A mutation. Altogether, the mutant probes works well as visualization tools to unearth the function of active sites in PIM-1 kinase, not only facilitating the further clarification of molecular mechanism underlying PIM-1 related signaling pathways, but also shedding light on drug development and disease therapy targeting PIM-1 protein.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 12","pages":"e202400443"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845612","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":"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}