Biotechnology Journal最新文献

{"title":"Miniaturization of an enclosed electrospinning process to enhance reproducibility in the fabrication of rapidly dissolving cell-based biosensors","authors":"Patrick Morkus,&nbsp;Stephanie Sibbald,&nbsp;Lauren Choi,&nbsp;Sarah Rassenberg,&nbsp;Carlos D. M. Filipe,&nbsp;David R. Latulippe","doi":"10.1002/biot.202300306","DOIUrl":"10.1002/biot.202300306","url":null,"abstract":"<p>There is broad interest in producing electrospun films embedded with biological materials. It is well known that electrospinning requires careful control of the process conditions, especially the environmental conditions such as relative humidity (RH). Given that commercial electrospinning systems are expensive (&gt; $10,000) and are typically too large to be used in standard biological safety cabinets (BSC), we designed and built a miniaturized electrospinning box (E-Box) that will fit inside a BSC, and the RH can be easily controlled using simple instrumentation (gas cylinder, regulator, needle valve, rotameter). It uses an inexpensive computerized numerical control machine to control the spinneret positioning and collector rotational speed—all the parts for the device (except the syringe pump and voltage supply) can be purchased for approximately $1000. We demonstrate the usefulness of our design in optimizing the production of <i>Escherichia coli</i>-embedded pullulan-trehalose films to be used as rapidly dissolving biosensors for environmental monitoring. At a fixed electrospinning recipe, we showed that decreasing the RH from approximately 48% to 22% resulted in the average fiber diameter increasing from 240 (± 11) nm to 314 (± 8) nm. We also demonstrate the usefulness of our design in performing sequential electrospinning experiments to evaluate process performance reproducibility. For example, from just 1 mL of a polymer solution, we produced 16 electrospun films (approximately 3 cm by 8 cm each)—from those films we hole-punched approximately 80 biosensor discs which were then used in subsequent experiments to determine the amount of two different biocides (Grotan BK and triclosan) in aqueous samples. The technique developed in this study is ideal for creating electrospun materials in high quantities that are highly reproducible through the precise control of RH.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300306","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50159980","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":"Advances in biosynthesis of peptide drugs: Technology and industrialization","authors":"Jing Wang,&nbsp;Long Chen,&nbsp;Song Qin,&nbsp;Mingyuan Xie,&nbsp;Shi-Zhong Luo,&nbsp;Wenjun Li","doi":"10.1002/biot.202300256","DOIUrl":"10.1002/biot.202300256","url":null,"abstract":"<p>Peptide drugs are developed from endogenous or synthetic peptides with specific biological activities. They have advantages of strong target specificity, high efficacy and low toxicity, thus showing great promise in the treatment of many diseases such as cancer, infections, and diabetes. Although an increasing number of peptide drugs have entered market in recent years, the preparation of peptide drug substances is yet a bottleneck problem for their industrial production. Comparing to the chemical synthesis method, peptide biosynthesis has advantages of simple synthesis, low cost, and low contamination. Therefore, the biosynthesis technology of peptide drugs has been widely used for manufacturing. Herein, we reviewed the development of peptide drugs and recent advances in peptide biosynthesis technology, in order to shed a light to the prospect of industrial production of peptide drugs based on biosynthesis technology.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54227106","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":"Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures","authors":"Shiaki A. Minami,&nbsp;Shruthi S. Garimella,&nbsp;Priya S. Shah","doi":"10.1002/biot.202300071","DOIUrl":"10.1002/biot.202300071","url":null,"abstract":"<p>Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300071","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50156601","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":"Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation","authors":"Augusto Costa-Barbosa,&nbsp;Diogo Ferreira,&nbsp;Maria Inês Pacheco,&nbsp;Margarida Casal,&nbsp;Henrique Oliveira Duarte,&nbsp;Catarina Gomes,&nbsp;Ana Margarida Barbosa,&nbsp;Egídio Torrado,&nbsp;Paula Sampaio,&nbsp;Tony Collins","doi":"10.1002/biot.202300219","DOIUrl":"10.1002/biot.202300219","url":null,"abstract":"<p>Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the <i>Candida albicans</i> cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in <i>Pichia pastoris</i> and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly <i>O</i>-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50156600","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 phenylalanine ammonia lyase to limit feedback inhibition by cinnamate and enhance biotransformation","authors":"Siddhi Pavale,&nbsp;Sudipt Kumar Dalei,&nbsp;Preeti Sokhal,&nbsp;Biswambhar Biswas,&nbsp;Kunal Meena,&nbsp;Nidhi Adlakha","doi":"10.1002/biot.202300275","DOIUrl":"10.1002/biot.202300275","url":null,"abstract":"<p>Phenylalanine ammonia-lyase (PAL) is a crucial enzyme for various biotechnology applications, such as producing phenols, antioxidants, and nutraceuticals. However, feedback inhibition from its product, cinnamic acid, limits its forward reaction rate. Therefore, this study aims to address the feedback inhibition in PAL using enzyme engineering strategies. Random and site-directed mutagenesis approaches were utilized to screen mutant enzymes with ameliorated tolerance against cinnamic acid. A thermotolerant and cinnamate-tolerant mutant was rationally identified using a high throughput screening method and subsequent biochemical characterization. We evaluated cinnamate affinity among the seven rationally selected mutations, and the T102E mutation was identified as the most promising mutant. This mutant showed a six-fold reduction in the affinity of PAL for cinnamic acid and a two-fold increase in operational stability compared with native PAL. Furthermore, the enzyme was immobilized on carbon nanotubes to increase its robustness and reusability. The immobilized mutant PAL showed greater efficiency in the deamination of phenylalanine present in protein hydrolysate than its free form. The rationale behind the enhancement of cinnamate tolerance was validated using molecular dynamic simulations. Overall, the knowledge of the sequence-function relationship of PAL was applied to drive enzyme engineering to develop highly tolerant PAL.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49671985","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":"Evaluation of expression systems for recombinant protein production in chicken egg bioreactors","authors":"Ryota Kajihara,&nbsp;Ryo Ezaki,&nbsp;Tenkai Watanabe,&nbsp;Kennosuke Ichikawa,&nbsp;Mei Matsuzaki,&nbsp;Hiroyuki Horiuchi","doi":"10.1002/biot.202300316","DOIUrl":"10.1002/biot.202300316","url":null,"abstract":"<p>Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5′ side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49671987","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":"Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece","authors":"Sohee Lim,&nbsp;Hyung-Jun Kwon,&nbsp;Dae Gwin Jeong,&nbsp;Hualin Nie,&nbsp;Sanghee Lee,&nbsp;Seo-Rin Ko,&nbsp;Kyu-Sun Lee,&nbsp;Young Bae Ryu,&nbsp;Hugh S. Mason,&nbsp;Hyun-Soon Kim,&nbsp;Ah-Young Shin,&nbsp;Suk-Yoon Kwon","doi":"10.1002/biot.202300319","DOIUrl":"10.1002/biot.202300319","url":null,"abstract":"<p>Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (μ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of μ-tp and KDEL to the ACE2 protein (ACE2 μK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 μK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 μK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300319","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49671986","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":"Peptonics: A new family of cell-protecting surfactants for the recombinant expression of therapeutic proteins in mammalian cell cultures","authors":"Ka Zhang,&nbsp;Eduardo Barbieri,&nbsp;Jacob LeBarre,&nbsp;Shahid Rameez,&nbsp;Sigma Mostafa,&nbsp;Stefano Menegatti","doi":"10.1002/biot.202300261","DOIUrl":"10.1002/biot.202300261","url":null,"abstract":"<p>Polymer surfactants are key components of cell culture media as they prevent mechanical damage during fermentation in stirred bioreactors. Among cell-protecting surfactants, Pluronics are widely utilized in biomanufacturing to ensure high cell viability and productivity. Monodispersity of monomer sequence and length is critical for the effectiveness of Pluronics—since minor deviations can damage the cells—but is challenging to achieve due to the stochastic nature of polymerization. Responding to this challenge, this study introduces Peptonics, a novel family of peptide and peptoid surfactants whose monomer composition and sequence are designed to achieve high cell viability and productivity at a fraction of chain length and cost of Pluronics. A designed ensemble of Peptonics was initially characterized via light scattering and tensiometry to select sequences whose phase behavior and tensioactivity align with those of Pluronics. Selected sequences were evaluated as cell-protecting surfactants using Chinese hamster ovary (CHO) cells expressing therapeutic monoclonal antibodies (mAb). Peptonics IH-T1010, ih-T1010, and ih-T1020 afforded high cell density (up to 3 × 10⁷ cells mL⁻¹) and viability (up to 95% within 10 days of culture), while reducing the accumulation of ammonia (a toxic metabolite) by ≈10% compared to Pluronic F-68. Improved cell viability afforded high mAb titer (up to 5.5 mg mL⁻¹) and extended the production window beyond 14 days; notably, Peptonic IH-T1020 decreased mAb fragmentation and aggregation ≈5%, and lowered the titer of host cell proteins by 16% compared to Pluronic F-68. These features can improve significantly the purification of mAbs, thus increasing their availability at a lower cost to patients.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300261","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41230817","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":"Regulatory mechanisms and cell membrane properties of Candida glycerinogenes differ under 2-phenylethanol addition or fermentation conditions","authors":"Yuqin Wang,&nbsp;Fang Liu,&nbsp;Xinyao Lu,&nbsp;Hong Zong,&nbsp;Bin Zhuge","doi":"10.1002/biot.202300181","DOIUrl":"10.1002/biot.202300181","url":null,"abstract":"<p>The biosynthesis of 2-phenylethanol (2-PE) at high yields and titers is often limited by its toxicity. In this study, we describe the molecular mechanisms of 2-PE tolerance in the multi-stress tolerant industrial yeast, <i>Candida glycerinogenes</i>. They were different under 2-PE addition or fermentation conditions. After extracellular addition of 2-PE, <i>C. glycerinogenes</i> cells became rounder and bigger, which reduced specific surface area. However, during 2-PE fermentation <i>C. glycerinogenes</i> cells were smaller, which increased specific surface area. Other differences in the tolerance mechanisms were studied by analyzing the composition and molecular parameters of the cell membrane. Extracellular 2-PE stress resulted in down-regulation of transcriptional expression of unsaturated fatty acid synthesis genes. This raised the proportion of saturated fatty acids in the cell membrane, which increased rigidity of the cell membrane and reduced 2-PE entry to the cell. However, intracellular 2-PE stress resulted in up-regulation of transcriptional expression of unsaturated fatty acid synthesis genes, and increased the proportion of unsaturated fatty acids in the cell membrane; this in turn enhanced flexibility of the cell membrane which accelerated efflux of 2-PE. These contrasting mechanisms are mediated by transcriptional factors Hog1 and Swi5. Under 2-PE addition, <i>C. glycerinogenes</i> activated Hog1 and repressed Swi5 to upregulate <i>erg5</i> and <i>erg4</i> expression, which increased cell membrane rigidity and resisted 2-PE import. During 2-PE fermentation, <i>C. glycerinogenes</i> activated Hog1 and repressed Swi5 to upregulate 2-PE transporter proteins <i>cdr1</i> and Acyl-CoA desaturase 1 <i>ole1</i> to increase 2-PE export, thus reducing 2-PE intracellular toxicity. The results provide new insights into 2-PE tolerance mechanisms at the cell membrane level and suggest a novel strategy to improve 2-PE production by engineering anti-stress genes.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41230818","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":"Aptamer-modified tetrahedral DNA nanostructure-immobilized liposome for specific gene delivery and potential cancer theragnostic","authors":"Doo Young Lim,&nbsp;Byeong Hee Hwang","doi":"10.1002/biot.202300156","DOIUrl":"10.1002/biot.202300156","url":null,"abstract":"<p>Targeted delivery of therapeutic agents to cancer cells is crucial for effective cancer treatment without adverse effects. In this study, we developed a novel delivery carrier, Aptamer-modified tetrahedral DNA nanostructure (TDN) immobilized Liposome (ApTL), for specific delivery to nucleolin-overexpressing cancer cells. We demonstrated that targeted ApTL was highly effective in delivering plasmid and mRNA to nucleolin-overexpressing cancer cells compared to non-targeted ApTL with a non-specific aptamer. ApTL, which is highly negative and nano-sized, specifically delivered nucleic acids to MDA-MB-231 and HeLa cancer cells, primarily via lipid-raft-mediated endocytosis. Furthermore, the co-delivery of mRNA and doxorubicin resulted in increased apoptosis and reduced cancer cell viability. Interestingly, co-delivery of mRNA and Dox did not show a significant difference in EGFP expression at 24 h but dramatically increased EGFP expression at 48 h, making ApTL/mEGFP/Dox a promising candidate for detecting live cancer cells after targeted cancer drug treatment. Our results suggest that ApTL can be a promising tool for the targeted delivery of therapeutic agents to nucleolin-overexpressing cancer cells, providing a new strategy for cancer theragnostic.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2023-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41186445","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}