Biotechnology Journal最新文献

{"title":"Physiological Metabolic Analysis of VB12 Accumulation in Ensifer adhaerens Casida A Enhanced by Oxygen Limitation","authors":"Bo Li,&nbsp;Xinyi Chen,&nbsp;Dujuan Zhao,&nbsp;Zebo Liu,&nbsp;Junming Li,&nbsp;Muhammad Safwan Siddique,&nbsp;Jiequn Wu,&nbsp;Yingping Zhuang,&nbsp;Zejian Wang","doi":"10.1002/biot.202400305","DOIUrl":"10.1002/biot.202400305","url":null,"abstract":"<div>\u0000 \u0000 <p>Cobalamin (VB₁₂) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB₁₂ biosynthesis by <i>Ensifer adhaerens Casida A</i> and has been identified as a bottleneck for economical substrate consumption. This study elucidates the relationship between oxygen limitation and VB₁₂ accumulation with transcriptomic and metabolomic analyses. Under oxygen limitation, <i>E. adhaerens</i> enhances oxygen transport and storage by increasing expression of flavin hemoglobin (<i>Hmp</i>), which was up-regulated 6-fold at 24 h of oxygen restriction compared to the oxygen restriction of 4 h (<i>p</i> &lt; 0.01). Because of the cofactor of <i>Hmp</i> is heme, the demand for heme increases, leading to the upregulation of genes in the heme biosynthesis pathway. Similarly, genes involved in biosynthesis of its precursor, 5-ALA, were upregulated as well. 5-ALA is also a direct precursor of VB₁₂, further leading to the upregulation of genes in the VB₁₂ biosynthesis pathway. This process initiates biosynthesis and accumulation of VB₁₂. As VB₁₂ and heme biosynthesis progresses, genes associated with the biosynthesis and transportation pathways of compounds related to their biosynthesis were likewise upregulated, including genes involved in S-adenosyl methionine (SAM) biosynthesis, and the transport of Fe²⁺ and Co²⁺. Additionally, amino acids and organic acids associated with biosynthesis were also extensively consumed, such as methionine, which is used for synthesizing SAM, decreased by 310% after 24 h of oxygen limitation compared to 20% dissolved oxygen (<i>p</i> &lt; 0.05). At the same time, genes related to growth-associated metabolic pathways, such as pentose phosphate pathway (PPP), were significantly downregulated. Therefore, the potential mechanism by which <i>E. adhaerens</i> accumulates VB₁₂ under oxygen-limited conditions by enhancing <i>Hmp</i> expression, which facilitates the porphyrin metabolic pathway and promotes VB₁₂ biosynthesis. This research provides valuable insights for increasing VB₁₂ production through metabolic engineering and process optimization.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256068","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":"Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production","authors":"Peter Andorfer,&nbsp;Carolin-Isabel Kahlig,&nbsp;Doris Pakusic,&nbsp;Robert Pachlinger,&nbsp;Christiane John,&nbsp;Irene Schrenk,&nbsp;Peter Eisenhut,&nbsp;Johannes Lengler,&nbsp;Bernd Innthaler,&nbsp;Lucia Micutkova,&nbsp;Barbara Kraus,&nbsp;Corey Brizzee,&nbsp;Jack Crawford,&nbsp;Juan A. Hernandez Bort","doi":"10.1002/biot.202400415","DOIUrl":"10.1002/biot.202400415","url":null,"abstract":"<p>In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400415","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142152667","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 9/2024","authors":"","doi":"10.1002/biot.202470091","DOIUrl":"https://doi.org/10.1002/biot.202470091","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470091","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165482","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":"Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems","authors":"Rafaela Meutelet,&nbsp;Lea J. Bisch,&nbsp;Benedikt C. Buerfent,&nbsp;Markus Müller,&nbsp;Jürgen Hubbuch","doi":"10.1002/biot.202400394","DOIUrl":"10.1002/biot.202400394","url":null,"abstract":"<p>The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400394","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142152668","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":"F-actin microfilaments affect the LIPUS-promoted osteogenic differentiation of BMSCs through TRPM7","authors":"Huan Yao,&nbsp;Li Tang,&nbsp;Dong Wang,&nbsp;Hua Pang,&nbsp;Ke Yang","doi":"10.1002/biot.202400310","DOIUrl":"https://doi.org/10.1002/biot.202400310","url":null,"abstract":"<p>The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of <i>Trpm7</i> mRNA. Moreover, adenoviral <i>Trpm7</i> knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of <i>Trpm7</i> suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100195","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 closed 3D printed microfluidic device for automated growth and differentiation of cerebral organoids from single-cell suspension","authors":"Mario Kandra,&nbsp;Tereza Vanova,&nbsp;Vincent A. Jongen,&nbsp;Jakub Pospíšil,&nbsp;Josef Novák,&nbsp;Václav Chochola,&nbsp;Tomáš Buryška,&nbsp;Zbyněk Prokop,&nbsp;Zdeněk Hodný,&nbsp;Ales Hampl,&nbsp;Dasa Bohaciakova,&nbsp;Josef Jaros","doi":"10.1002/biot.202400240","DOIUrl":"https://doi.org/10.1002/biot.202400240","url":null,"abstract":"<p>The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100198","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 3D bioprinted antibacterial hydrogel dressing of gelatin/sodium alginate loaded with ciprofloxacin hydrochloride","authors":"Liuyuan Cao,&nbsp;Yueqi Lu,&nbsp;Hezhi Chen,&nbsp;Ya Su,&nbsp;YuneYee Cheng,&nbsp;Jie Xu,&nbsp;Huanwei Sun,&nbsp;Kedong Song","doi":"10.1002/biot.202400209","DOIUrl":"https://doi.org/10.1002/biot.202400209","url":null,"abstract":"<p>Skin plays a crucial role in human physiological functions, however, it was vulnerable to bacterial infection which delayed wound healing. Nowadays, designing an individual wound dressing with good biocompatibility and sustaining anti-infection capability for healing of chronic wounds are still challenging. In this study, various concentrations of the ciprofloxacin (CIP) were mixed with gelatine (Gel)/sodium alginate (SA) solution to prepare Gel/SA/CIP (GAC) bioinks, following the fabrication of GAC scaffold by an extrusion 3D bioprinting technology. The results showed that the GAC bioinks had good printability and the printed GAC scaffolds double-crosslinked by EDC/NHS and CaCl₂ had rich porous structure with appropriate pore size, which were conducive to drug release and cell growth. It demonstrated that the CIP could be rapidly released by 70% in 5 min, which endowed the GAC composite scaffolds with an excellent antibacterial ability. Especially, the antibacterial activities of GAC7.5 against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> within 24 h were even close to 100%, and the inhibition zones were still maintained 14.78 ± 0.40 mm and 14.78 ± 0.40 mm, respectively, after 24 h. Meanwhile, GAC7.5 also demonstrated impressive biocompatibility which can promote the growth and migration of L929 and accelerate wound healing. Overall, the GAC7.5 3D bioprinting scaffold could be used as a potential skin dressing for susceptible wounds with excellent antibacterial activity and good biocompatibility to meet urgent clinical needs.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100197","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":"Bicarbonate concentration influences carbon utilization rates and biochemical profiles of freshwater and marine microalgae","authors":"Philip Asare Kusi,&nbsp;Donal McGee,&nbsp;Shamas Tabraiz,&nbsp;Asma Ahmed","doi":"10.1002/biot.202400361","DOIUrl":"https://doi.org/10.1002/biot.202400361","url":null,"abstract":"<p>Selecting the optimal microalgal strain for carbon capture and biomass production is crucial for ensuring the commercial viability of microalgae-based biorefinery processes. This study aimed to evaluate the impact of varying bicarbonate concentrations on the growth rates, inorganic carbon (IC) utilization, and biochemical composition of three freshwater and two marine microalgal species. <i>Parachlorella kessleri</i>, <i>Vischeria</i> cf. <i>stellata</i>, and <i>Porphyridium purpureum</i> achieved the highest carbon removal efficiency (&gt;85%) and biomass production at 6 g L⁻¹ sodium bicarbonate (NaHCO₃), while <i>Phaeodactylum tricornutum</i> showed optimal performance at 1 g L⁻¹ NaHCO₃. The growth and carbon removal rate of <i>Scenedesmus quadricauda</i> increased with increasing NaHCO₃ concentrations, although its highest carbon removal efficiency (∼70%) was lower than the other species. Varying NaHCO₃ levels significantly impacted the biochemical composition of <i>P. kessleri</i>, <i>S. quadricauda</i>, and <i>P. purpureum</i> but did not affect the composition of the remaining species. The fatty acid profiles of the microalgae were dominated by C16 and C18 fatty acids, with <i>P. purpureum</i> and <i>P. tricornutum</i> yielding relatively high polyunsaturated fatty acid content ranging between 14% and 30%. Furthermore, bicarbonate concentration had a species-specific effect on the fatty acid and chlorophyll-<i>a</i> content. This study demonstrates the potential of bicarbonate as an effective IC source for microalgal cultivation, highlighting its ability to select microalgal species for various applications based on their carbon capture efficiency and biochemical composition.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400361","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100199","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":"Characterizing interactions of endoplasmic reticulum resident proteins in situ through the YST-PPI method","authors":"Xian Fan,&nbsp;Huahua He,&nbsp;Ting Wang,&nbsp;Pan Xu,&nbsp;Faying Zhang,&nbsp;Shantong Hu,&nbsp;Yueli Yun,&nbsp;Meng Mei,&nbsp;Guimin Zhang,&nbsp;Li Yi","doi":"10.1002/biot.202400346","DOIUrl":"https://doi.org/10.1002/biot.202400346","url":null,"abstract":"<p>The mutual interactions of endoplasmic reticulum (ER) resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease system for Protein-Protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology, and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER-resident proteins, most being identified molecular chaperones, of <i>S. cerevisiae</i> were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER-resident proteins, except the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER-resident proteins perform their functions in a cooperative way and a multimer complex might be formed during the process.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100194","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":"Dialysis rolled scaffold bioreactor allows extended production of monoclonal antibody with reduced media use","authors":"Tongyao Wu,&nbsp;Mohsen Norouzi,&nbsp;Kidong Park","doi":"10.1002/biot.202400249","DOIUrl":"https://doi.org/10.1002/biot.202400249","url":null,"abstract":"<p>Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142100193","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}