Maximilian Klimpel, Monica Terrao, Melina Bräuer, Herbert Dersch, Martina Biserni, Larissa Melo Do Nascimento, Sarah Schwingal, Jessica E. Vogel, Cathrin Ferlemann, Tobias Brandt, Nikki Indresh Lal, Krystal Bridgeman, Alex Petzke, Eva McDwyer, Jo Leen Lim, Seungyoul Oh, Gabriela Brumatti, Albert Garcia Minambres, Ellen Otte, Thomas Noll, Vicky Pirzas, Holger Laux
The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL−1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL−1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL−1 day−1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.
用水泡性口炎病毒包膜糖蛋白(VSV-G)伪型慢病毒载体(LV)的生产受到了包膜相关细胞毒性和生产方法(如瞬时转染粘附细胞系)的限制。在这项研究中,我们建立了稳定的悬浮生产细胞系,用于规模化和无血清生产 LV,这些细胞系来自两个稳定的诱导包装细胞系,分别命名为 GPRG 和 GPRTG。已建立的多克隆生产者细胞系可在摇瓶中以半灌流方式生产携带 WAS-T2A-GFP 构建体的自失活(SIN)LV,平均感染滴度高达 4.64 × 107 TU mL-1,且可在两个月内生成。衍生的单克隆细胞系在连续培养过程中功能稳定,在半灌注摇瓶过程中产生的平均感染滴度高达 9.38 × 107 TU mL-1。在非优化的 5 L 搅拌罐生物反应器灌注工艺中,生产克隆能够连续 29 天保持 >1 × 107 TU mL-1 的生产率,这是 LV 生产领域的一个重要里程碑。由于生产细胞系基于可诱导的 Tet-off 表达系统,所建立的工艺可在没有抗生素等诱导剂的情况下生产 LV。纯化的 LV 能有效转导人类 CD34+ 细胞,减少基因和细胞疗法应用所需的 LV 数量。
{"title":"Generation of stable suspension producer cell lines for serum-free lentivirus production","authors":"Maximilian Klimpel, Monica Terrao, Melina Bräuer, Herbert Dersch, Martina Biserni, Larissa Melo Do Nascimento, Sarah Schwingal, Jessica E. Vogel, Cathrin Ferlemann, Tobias Brandt, Nikki Indresh Lal, Krystal Bridgeman, Alex Petzke, Eva McDwyer, Jo Leen Lim, Seungyoul Oh, Gabriela Brumatti, Albert Garcia Minambres, Ellen Otte, Thomas Noll, Vicky Pirzas, Holger Laux","doi":"10.1002/biot.202400090","DOIUrl":"10.1002/biot.202400090","url":null,"abstract":"<p>The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 10<sup>7</sup> TU mL<sup>−1</sup> in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 10<sup>7</sup> TU mL<sup>−1</sup> in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 10<sup>7</sup> TU mL<sup>−1</sup> day<sup>−1</sup> for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34<sup>+</sup> cells, reducing the LV quantities required for gene and cell therapy applications.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400090","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891383","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}
The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of β-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.
抗生素的发现极大地促进了人类文明的发展,然而,滥用和过度使用抗生素所导致的细菌耐药性却极大地挑战着人类健康和食品安全。面对日益严峻的形势,人们迫切要求开发新兴的非传统技术或方法来解决这一问题。随着合成生物学的发展,光遗传学在许多领域的基因表达精确调控方面展现出诱人的前景。因此,利用光遗传学来降低抗生素耐药性的风险很有吸引力。本文基于一种光敏 DNA 结合蛋白(EL222),在大肠杆菌中建立了一种蓝光可控基因表达系统。此外,还利用蓝光成功抑制了β-内酰胺酶基因(bla)的表达,从而大大降低了工程大肠杆菌对氨苄西林的耐药性。此外,利用蓝光诱导大电导机械敏感通道(MscL)的表达,成功地提高了工程大肠杆菌对链霉素的敏感性。最后,在同一个大肠杆菌细胞中,蓝光同时诱导氨苄西林和链霉素敏感性的增加,揭示了这一策略在控制耐多药(MDR)细菌方面的巨大潜力。作为概念验证,我们的工作证明了光可以作为一种替代工具,在不开发新抗生素的情况下延长普通抗生素的使用期。这种基于光遗传学的新策略为未来抗击抗生素耐药性开辟了前景广阔的道路。
{"title":"Blue light-mediated gene expression as a promising strategy to reduce antibiotic resistance in Escherichia coli","authors":"Qingwei Jiang, Feng Geng, Juan Shen, Ping Zhu, Zhaoxin Lu, Fengxia Lu, Libang Zhou","doi":"10.1002/biot.202400023","DOIUrl":"10.1002/biot.202400023","url":null,"abstract":"<p>The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in <i>Escherichia coli</i> based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of β-lactamase gene (<i>bla</i>) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered <i>E. coli</i>. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered <i>E. coli</i>. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same <i>E. coli</i> cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891297","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}
CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol−1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min−1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production—herein detected for the first time as CYP116B5 metabolite—compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.
{"title":"CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation","authors":"Daniele Giuriato, Gianluca Catucci, Danilo Correddu, Giovanna Di Nardo, Gianfranco Gilardi","doi":"10.1002/biot.202300664","DOIUrl":"10.1002/biot.202300664","url":null,"abstract":"<p>CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H<sub>2</sub>O<sub>2</sub>. Recently, the <i>Molecular Lego</i> approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H<sub>2</sub>O<sub>2</sub> in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a T<sub>M</sub> of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol<sup>−1</sup> for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H<sub>2</sub>O<sub>2</sub> (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H<sub>2</sub>O<sub>2</sub> generation, an improved <i>k<sub>cat</sub>/K<sub>M</sub></i> for the <i>p</i>-nitrophenol conversion was observed (<i>k<sub>cat</sub></i> of 20.1 ± 0.6 min<sup>−1</sup> and <i>K<sub>M</sub></i> of 0.23 ± 0.03 mM), corresponding to 4 times the <i>k<sub>cat</sub>/K<sub>M</sub></i> of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an <i>E. coli</i> strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen <i>N</i>-oxide production—herein detected for the first time as CYP116B5 metabolite—compared to the direct H<sub>2</sub>O<sub>2</sub> supply, equal to the 25% of the total drug conversion.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891335","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}
Thi Kieu Oanh Nguyen, Dayoung Ryu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Thi Luong Vu, Han Choe
Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.
{"title":"Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain","authors":"Thi Kieu Oanh Nguyen, Dayoung Ryu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Thi Luong Vu, Han Choe","doi":"10.1002/biot.202300581","DOIUrl":"10.1002/biot.202300581","url":null,"abstract":"<p>Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in <i>Escherichia coli</i> has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various <i>E. coli</i> strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300581","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891414","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}
Carolin-Isabel Kahlig, Sylvain Moser, Lucia Micutkova, Johannes Grillari, Barbara Kraus, Juan A. Hernandez Bort
The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields.
In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines.
Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.
{"title":"Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures","authors":"Carolin-Isabel Kahlig, Sylvain Moser, Lucia Micutkova, Johannes Grillari, Barbara Kraus, Juan A. Hernandez Bort","doi":"10.1002/biot.202300672","DOIUrl":"10.1002/biot.202300672","url":null,"abstract":"<p>The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields.</p><p>In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines.</p><p>Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300672","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891420","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}
Edgar Trujillo, Elizabeth Monreal-Escalante, Carlos Angulo
Microalgal emergence is a promising platform with two-decade historical background for producing vaccines and biopharmaceuticals. During that period, microalgal-based vaccines have reported successful production for various diseases. Thus, species selection is important for genetic transformation and delivery methods that have been developed. Although many vaccine prototypes have been produced for infectious and non-infectious diseases, fewer studies have reached immunological and immunoprotective evaluations. Microalgae-made vaccines for Staphylococcus aureus, malaria, influenza, human papilloma, and Zika viruses have been explored in their capacity to induce humoral or cellular immune responses and protective efficacies against experimental challenges. Therefore, specific pathogen antigens and immune system role are important and addressed in controlling these infections. Regarding non-communicable diseases, these vaccines have been investigated for breast cancer; microalgal-produced therapeutic molecules and microalgal-made interferon-α have been explored for hypertension and potential applications in treating viral infections and cancer, respectively. Thus, conducting immunological trials is emphasized, discussing the promising results observed in terms of immunogenicity, desired immune response for controlling affections, and challenges for achieving the desired protection levels. The potential advantages and hurdles associated with this innovative approach are highlighted, underlining the relevance of assessing immune responses in preclinical and clinical trials to validate the efficacy of these biopharmaceuticals. The promising future of this healthcare technology is also envisaged.
{"title":"Microalgae-made human vaccines and therapeutics: A decade of advances","authors":"Edgar Trujillo, Elizabeth Monreal-Escalante, Carlos Angulo","doi":"10.1002/biot.202400091","DOIUrl":"10.1002/biot.202400091","url":null,"abstract":"<p>Microalgal emergence is a promising platform with two-decade historical background for producing vaccines and biopharmaceuticals. During that period, microalgal-based vaccines have reported successful production for various diseases. Thus, species selection is important for genetic transformation and delivery methods that have been developed. Although many vaccine prototypes have been produced for infectious and non-infectious diseases, fewer studies have reached immunological and immunoprotective evaluations. Microalgae-made vaccines for <i>Staphylococcus aureus</i>, malaria, influenza, human papilloma, and Zika viruses have been explored in their capacity to induce humoral or cellular immune responses and protective efficacies against experimental challenges. Therefore, specific pathogen antigens and immune system role are important and addressed in controlling these infections. Regarding non-communicable diseases, these vaccines have been investigated for breast cancer; microalgal-produced therapeutic molecules and microalgal-made interferon-α have been explored for hypertension and potential applications in treating viral infections and cancer, respectively. Thus, conducting immunological trials is emphasized, discussing the promising results observed in terms of immunogenicity, desired immune response for controlling affections, and challenges for achieving the desired protection levels. The potential advantages and hurdles associated with this innovative approach are highlighted, underlining the relevance of assessing immune responses in preclinical and clinical trials to validate the efficacy of these biopharmaceuticals. The promising future of this healthcare technology is also envisaged.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891434","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}
Young Sik Kim, Jung Soo Lee, Mi Yeong Jeong, Ju Woong Jang, Moon Suk Kim
Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.
{"title":"Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing","authors":"Young Sik Kim, Jung Soo Lee, Mi Yeong Jeong, Ju Woong Jang, Moon Suk Kim","doi":"10.1002/biot.202300596","DOIUrl":"10.1002/biot.202300596","url":null,"abstract":"<p>Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891448","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}
Mingyu Li, Xiaopeng Ren, Ming Xu, Sitong Dong, Xianzhen Li, Xiaoyi Chen, Conggang Wang, Fan Yang
Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.
{"title":"Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production","authors":"Mingyu Li, Xiaopeng Ren, Ming Xu, Sitong Dong, Xianzhen Li, Xiaoyi Chen, Conggang Wang, Fan Yang","doi":"10.1002/biot.202400178","DOIUrl":"10.1002/biot.202400178","url":null,"abstract":"<p>Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered <i>Bacillus subtilis</i> strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the <i>B. subtilis</i> strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL<sup>-1</sup> in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL<sup>-1</sup> during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered <i>B. subtilis</i> strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891304","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}
Jiwon Na, Ali Behboudi, Jiwon Mun, Hoeun Jin, Andrew L. Zydney, Youngbin Baek
Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.
{"title":"Protein loss during membrane processes in biopharmaceutical manufacturing","authors":"Jiwon Na, Ali Behboudi, Jiwon Mun, Hoeun Jin, Andrew L. Zydney, Youngbin Baek","doi":"10.1002/biot.202400154","DOIUrl":"10.1002/biot.202400154","url":null,"abstract":"<p>Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891440","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}
Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.
将生物元素自组装成生物仿生货物载体以进行靶向和输送是一种很有前景的方法。然而,它仍然面临着实际挑战。我们开发了一种用神经元生长因子(NGF)操纵神经元系统的 DNA 折纸(DO)纳米结构功能化方法。NGF 的生物活性及其与神经元系统的相互作用已在体外和体内模型中得到证实。通过分子自组装制造的 DO 元件通过向细胞表面受体静态呈现配体的空间和时间控制来操纵周围环境。我们的数据显示,它在体外分化 PC12 细胞方面具有有效的生物活性。此外,DNA 折纸 NGF (DON) 通过沿功能化 DO 结构梯度引入趋化效应,影响了培养中背根神经节神经元的生长方向性和空间能力。最后,我们发现这些元素在大鼠坐骨神经损伤模型中增强了轴突再生能力。这项研究证明了 DO 在神经元操作和再生中的功能。这里提出的方法,即由 DO 构建的可编程纳米级元件形成的工程平台,可以扩展到神经系统之外,并为再生医学、组织工程和细胞生物学领域带来革命性的变化。
{"title":"DNA origami scaffold promoting nerve guidance and regeneration","authors":"Jonathan Giron, Merav Antman-Passig, Neta Zilony, Hadas Schori, Ido Bachelet, Orit Shefi","doi":"10.1002/biot.202300734","DOIUrl":"10.1002/biot.202300734","url":null,"abstract":"<p>Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300734","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891342","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}