Biotechnology Journal最新文献

tsRNA-12391-Modified Adipose Mesenchymal Stem Cell-Derived Exosomes Mitigate Cartilage Degeneration in Osteoarthritis by Enhancing Mitophagy

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-04-03 DOI: 10.1002/biot.202400611

Jingsheng Shi, Guanglei Zhao, Siqun Wang, Yibing Wei, Jianguo Wu, Gangyong Huang, Jie Chen, Jun Xia

{"title":"tsRNA-12391-Modified Adipose Mesenchymal Stem Cell-Derived Exosomes Mitigate Cartilage Degeneration in Osteoarthritis by Enhancing Mitophagy","authors":"Jingsheng Shi, Guanglei Zhao, Siqun Wang, Yibing Wei, Jianguo Wu, Gangyong Huang, Jie Chen, Jun Xia","doi":"10.1002/biot.202400611","DOIUrl":"https://doi.org/10.1002/biot.202400611","url":null,"abstract":"<div>\u0000 \u0000 Osteoarthritis (OA) is a cartilage-degenerative joint disease. Mitophagy impacts articular cartilage damage. tRNA-derived small RNAs (tsRNAs) are one of the contents of adipose mesenchymal stem cell (AMSC)-derived exosomes (AMSC-exos) and are involved in disease progression. However, whether tsRNAs regulate mitophagy and whether tsRNA-modified AMSC-exos improve OA via mitophagy remain unclear. We performed small RNA sequencing to identify OA-related tsRNAs, which were then loaded into AMSC-exos, exploring the function and mechanisms related to mitophagy in vitro and in vivo. Overall, 53 differentially expressed tsRNAs (DEtsRNAs) were identified between OA and normal cartilage tissues, among which 42 DEtsRNAs, including tsRNA-12391, were downregulated in the OA group. Target genes of tsRNA-12391 mainly participated in mitophagy-related pathways such as Rap1 signaling pathway. Compared to the control group, tsRNA-12391 mimics significantly promoted mitophagy, as shown by the upregulated expression of PINK1 and LC3 and the co-localization of Mito-Tracker Green and PINK1. Furthermore, tsRNA-12391 mimics effectively enhanced chondrogenesis in chondrocytes, as demonstrated by the elevated expression of collagen II and ACAN. AMSC-exos with tsRNA-12391 overexpression also facilitated mitophagy and chondrogenesis in vitro and in vivo. Mechanistically, tsRNA-12391 bound to ATAD3A restricted ATAD31 from degrading PINK1, leading to PINK1 accumulation. ATAD31 overexpression reversed the effects of tsRNA-12391 mimics on mitophagy and chondrogenesis. AMSC-exos loaded with tsRNA-12391 promoted mitophagy and chondrogenesis by interacting with ATAD31; this may be a novel therapeutic strategy for OA.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762103","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}

引用次数: 0

Phage Display Selected Chicken Antibodies Targeting Surface Alpha Enolase in Staphylococcus aureus

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-04-01 DOI: 10.1002/biot.70011

Wei-Chu Wang, Chi-Hsin Lee, Chao-Jung Wu, Sy-Jye Leu, Pei-Shih Kao, Bor-Yu Tsai, Ko-Jiunn Liu, Yu-Wei Chiang, Hsiu-Jung Lo, Yan-Chiao Mao, Yi-Yuan Yang

{"title":"Phage Display Selected Chicken Antibodies Targeting Surface Alpha Enolase in Staphylococcus aureus","authors":"Wei-Chu Wang, Chi-Hsin Lee, Chao-Jung Wu, Sy-Jye Leu, Pei-Shih Kao, Bor-Yu Tsai, Ko-Jiunn Liu, Yu-Wei Chiang, Hsiu-Jung Lo, Yan-Chiao Mao, Yi-Yuan Yang","doi":"10.1002/biot.70011","DOIUrl":"https://doi.org/10.1002/biot.70011","url":null,"abstract":"<div>\u0000 \u0000 Staphylococcus aureus, a prevalent gram-positive bacterium in human populations, poses a significant risk for causing serious opportunistic infections and increasing antibiotic resistance. Alpha-enolase in S. aureus plays important roles in extracellular matrix binding and biofilm formation. These functions enable S. aureus to invade host tissues and cause infections. The aim of this study was to develop specific alpha-enolase chicken antibodies through phage display technology targeting S. aureus surface proteins as a potential alternative to antibiotic therapy. A chicken was immunized with recombinant S. aureus alpha-enolase, leading to the construction of two phage display single-chain variable fragment libraries of 3.32 × 106 and 8.60×105 transformants with different linker lengths. After four rounds of biopanning, five single-chain variable fragment antibody clones, including three with high binding affinities (SaS1, SaS2, and SaL2), were selected. These clones exhibited distinct binding patterns in epitope mapping and cross-reaction assays, with SaS1 and SaS2 specifically recognizing S. aureus alpha-enolase and SaL2 cross-reacting with Streptococcus pneumoniae alpha-enolase. Furthermore, the specificity of these antibody clones toward clinical S. aureus strains, including methicillin-sensitive and methicillin-resistant strains, was validated through cell-based enzyme-linked immunosorbent assays (ELISA) and flow cytometry assays. The identification of SaS1, SaS2, and SaL2 underscores their diagnostic and therapeutic potential, offering promising alternatives to traditional antibiotic therapies.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143740943","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}

引用次数: 0

Resolving the Trade-Off Between Toxicity and Efficiency of CRISPR-Cas9 System for Genome Editing Within Escherichia coli

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-04-01 DOI: 10.1002/biot.70010

Qian Guo, Qi Shen, Qi Hao, Xian-Long Jiang, Lu-Ping Zou, Ya-Ping Xue, Yu-Guo Zheng

{"title":"Resolving the Trade-Off Between Toxicity and Efficiency of CRISPR-Cas9 System for Genome Editing Within Escherichia coli","authors":"Qian Guo, Qi Shen, Qi Hao, Xian-Long Jiang, Lu-Ping Zou, Ya-Ping Xue, Yu-Guo Zheng","doi":"10.1002/biot.70010","DOIUrl":"https://doi.org/10.1002/biot.70010","url":null,"abstract":"<div>\u0000 \u0000 Efficient gene editing of Escherichia coli BL21 (DE3) holds significant practical value as a host for heterologous protein expression. Recently reported CRISPR-Cas9 editing systems for this strain exhibit a trade-off between efficiency and toxicity. In this study, we addressed this trade-off by employing the strategy to transiently induce Cas9 expression in the high-copy plasmid during the editing stage. Furthermore, we demonstrated that eliminating the sgRNA-expressing plasmid using a temperature-sensitive replicon, combined with SacB for removing the Cas9-expressing plasmid, exhibited higher efficiency compared to previously reported strategies for editing system removal. We assigned this optimized CRISPR-Cas9 genome editing system as the pEBcas9/pEBsgRNA system, which has successfully achieved efficient five rounds of genome editing and simultaneous editing of multiple loci in E. coli BL21 (DE3). Using this system, we identified several loci suitable for multi-copy integrated expression of exogenous genes. Overall, the pEBcas9/pEBsgRNA system may facilitate the application of E. coli in both industrial and academic fields.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 4","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143740940","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}

引用次数: 0

Issue Information: Biotechnology Journal 4/2025

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-04-01 DOI: 10.1002/biot.70016

引用次数: 0

Subcellular Fractionation Enables Assessment of Nucleotide Sugar Donors Inside the Golgi Apparatus as a Prerequisite for Unraveling Culture Impacts on Glycoforms of Antibodies

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-24 DOI: 10.1002/biot.202400678

Niklas Regett, Marcel Dieterle, Fleur Peters, Max Deuring, Kaja Stegmaier, Attila Teleki, Ralf Takors

{"title":"Subcellular Fractionation Enables Assessment of Nucleotide Sugar Donors Inside the Golgi Apparatus as a Prerequisite for Unraveling Culture Impacts on Glycoforms of Antibodies","authors":"Niklas Regett, Marcel Dieterle, Fleur Peters, Max Deuring, Kaja Stegmaier, Attila Teleki, Ralf Takors","doi":"10.1002/biot.202400678","DOIUrl":"https://doi.org/10.1002/biot.202400678","url":null,"abstract":"Glycosylation is a critical quality attribute in biopharmaceuticals that influences crucial properties, such as biological activity and blood clearance. Current methods for modeling glycosylation typically rely on imprecise or limited data on nucleotide sugar donor (NSD) dynamics. These methods use in vitro transporter kinetics or flux balance analysis, which overlook the key aspects of metabolic regulation. We devised an integrative workflow for absolute subcellular NSD quantification in both cytoplasm and secretory organelles. Using subcellular fractionation, exhaustive sample extraction, and liquid chromatography triple-quadrupole tandem mass spectrometry, we accurately measured NSD concentrations ranging from 1.6 amol/cell to 3 fmol/cell.As expected, NSD concentration profiles aligned closely with the glycan distributions on antibodies, particularly after nutrient pulsing to stimulate NSD production, showcasing method validity. This method enables empirical observation of compartment-specific NSD dynamics. Thus, this study provides novel insights indicating that N-glycosylation, which governs NSD supply, is primarily regulated within the Golgi apparatus (GA). This method offers a novel tool to obtain sophisticated data for a more efficient optimization of glycosylation processes in production cell lines.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400678","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690108","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}

引用次数: 0

Biodegradable Covalent Beta-Cyclodextrin Nanocages for Acidic and Reductive-Responsive Drug Delivery in Enhanced Tumor Therapy

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-24 DOI: 10.1002/biot.70006

Jingyi Xiao, Zan Ge, Xiaowei Tan, Ziyi Liu, Yafang Zhang, Shufen Xiao, Rongyuan Yi, Ye Hu, Wenyan Hu, Hui Chu, Jian Chen

{"title":"Biodegradable Covalent Beta-Cyclodextrin Nanocages for Acidic and Reductive-Responsive Drug Delivery in Enhanced Tumor Therapy","authors":"Jingyi Xiao, Zan Ge, Xiaowei Tan, Ziyi Liu, Yafang Zhang, Shufen Xiao, Rongyuan Yi, Ye Hu, Wenyan Hu, Hui Chu, Jian Chen","doi":"10.1002/biot.70006","DOIUrl":"https://doi.org/10.1002/biot.70006","url":null,"abstract":"<div>\u0000 \u0000 Traditional beta-cyclodextrin (beta-CD) in biomedical applications faces challenges due to its inherent physical and biochemical limitations. One of the most effective strategies to enhance the properties of beta-CD for drug delivery is the synthesis of supramolecular polycyclodextrins. In this study, we designed a novel beta-CD nanocage-like structure for drug delivery, incorporating imine and disulfide bonds through Schiff base reactions. Aldehyde group-functionalized beta-CD units were used to construct the main backbone of the nanocage, forming dual-dynamic covalent bonds. The chemical structure of the beta-CD nanocage was confirmed using ¹H nuclear magnetic resonance (¹H NMR) and Fourier transform infrared spectroscopy (FTIR). Additionally, atomic force microscopy (AFM) and dynamic light scattering (DLS) revealed that varying amounts of beta-CD crosslinked with cystamine resulted in nanocages approximately 200 nm in size. In vitro drug release experiments demonstrated that doxorubicin (DOX)-loaded beta-CD nanocages exhibited accelerated DOX release in acidic and reductive environments compared to normal physiological conditions, owing to the pH-sensitive imine bond and the glutathione (GSH)-cleavable disulfide bond. The DOX-loaded beta-CD nanocages showed exceptional tumor-killing effects, particularly in acid/reduction-enhanced tumor cells. Both cellular fluorescence imaging and flow cytometry confirmed the potential of the beta-CD nanocages for acid/reduction-specific drug release. Consequently, this precision medicine model using imine/disulfide-linked beta-CD nanocage structures as acidity/reduction-sensitive drug carriers promises to improve oncotherapy through more targeted drug delivery and release, supporting individualized treatment approaches.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690109","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}

引用次数: 0

Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-20 DOI: 10.1002/biot.70005

Xiaojie Zhou, Siqi Yang, Bingbing Sun, Feng Dong, Mingyu Yin, Yu Jiang, Zhiwei Huang, Sheng Yang

{"title":"Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli","authors":"Xiaojie Zhou, Siqi Yang, Bingbing Sun, Feng Dong, Mingyu Yin, Yu Jiang, Zhiwei Huang, Sheng Yang","doi":"10.1002/biot.70005","DOIUrl":"10.1002/biot.70005","url":null,"abstract":"<div>\u0000 \u0000 The preferred method for Escherichia coli genome editing relies on Cas9 from Streptococcus pyogenes (SpCas9) and λ-Red recombinase. Although SpCas9 is currently the most active RNA-guided DNA endonuclease, a significant number of escapers are often observed, making it inefficient across different sites, particularly when inserting large fragments. In this study, we identified two RAGATH RNA-associated DNA endonucleases (RADs) derived from IS607 transposons. Both of them exhibited high cleavage activity in E. coli. When combined with λ-Red recombinase, they achieved editing efficiencies approaching 100%. Even at target sites where SpCas9 exhibited low editing efficiency, RADs maintained efficiencies ranging from 57% to 94%. Moreover, RADs exhibited higher efficiencies in inserting large fragments in certain cases compared to SpCas9. Taken together, these RAD-based genome editing tools provide viable alternatives to SpCas9, particularly for challenging targets and/or large fragment insertions.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143661649","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}

引用次数: 0

Rapid At-Line AAVX Affinity HPLC: Enabling Process Analytical Technology for Bioprocess Development of Adeno-Associated Virus Vectors

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-18 DOI: 10.1002/biot.202400656

Jakob Heckel, Timo Bohlig, Lea Bonnington, Michael Leiss, Markus Haindl, Jürgen Hubbuch, Tobias Graf

{"title":"Rapid At-Line AAVX Affinity HPLC: Enabling Process Analytical Technology for Bioprocess Development of Adeno-Associated Virus Vectors","authors":"Jakob Heckel, Timo Bohlig, Lea Bonnington, Michael Leiss, Markus Haindl, Jürgen Hubbuch, Tobias Graf","doi":"10.1002/biot.202400656","DOIUrl":"https://doi.org/10.1002/biot.202400656","url":null,"abstract":"Recombinant adeno-associated virus (rAAV) vectors have emerged as a new class of therapeutic modal with the promise to treat or even cure hereditary and acquired diseases, but their consistent and efficient production remains challenging. To address these inadequacies, the implementation of process analytical technology (PAT) principles for the development of rAAV-based gene therapies holds the prospect of promoting greater product and process understanding. However, a substantial lack of suitable analytical tools during both upstream and downstream processing (DSP) hinders the ability to fully realize the potential of PAT for rAAVs. To fill this gap, our recently described AAVX affinity-based high-performance liquid chromatography (HPLC) method was assessed as an at-line PAT tool to determine the capsid titer and the percentage of filled capsids at various stages of the production process. Leveraging the fast and robust provision of these parameters, even for challenging samples, the benefits of this approach for improved process monitoring and control were demonstrated for samples generated both during fermentation and DSP. Given the versatility of our developed analytical method for different rAAV serotype and payload combinations, we eventually highlight its expansive opportunities to streamline process development and therefore contributing to high-quality and cost-efficient production of rAAV-based gene therapies.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400656","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143645695","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}

引用次数: 0

Study and Application of Microbial Community in Biodegradation of Kitchen Waste Using High-Throughput Sequencing and Strain Isolation Techniques

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-18 DOI: 10.1002/biot.70001

Yingshuai Wu, Han Zhao, Yuanwei Wang, Shitong Dong, Ruiming Wang, Chunling Ma

{"title":"Study and Application of Microbial Community in Biodegradation of Kitchen Waste Using High-Throughput Sequencing and Strain Isolation Techniques","authors":"Yingshuai Wu, Han Zhao, Yuanwei Wang, Shitong Dong, Ruiming Wang, Chunling Ma","doi":"10.1002/biot.70001","DOIUrl":"https://doi.org/10.1002/biot.70001","url":null,"abstract":"<div>\u0000 \u0000 Kitchen waste contains abundant starch, cellulose, lignin, and other organic compounds. This study investigated the microbial diversity of composite microbial communities capable of efficiently degrading kitchen waste, with a focus on the isolation of lignin-degrading strains. High-throughput sequencing on the Illumina MiSeq platform was utilized to analyze the diversity and community structure of bacteria and fungi in kitchen waste, enabling the identification of core microbiota. Approximately 60 strains, representing over 20 genera, were successfully isolated and identified using pure culture techniques. Comparative analysis of microbial diversity, based on high-throughput sequencing and traditional strain isolation methods, revealed that bacteria predominantly belonged to the phylum Firmicutes, while fungi were mainly classified into Ascomycota and Basidiomycota. These methods provided complementary insights into the composition and diversity of microbial communities. Among the culturable strains, three high-yield laccase-producing fungi—Schizophyllum commune, Alternaria alternata, and Coriolopsis trogii—were selected. The laccase genes Sc-lac and Aa-lac were synthesized and heterologously expressed in Pichia pastoris, resulting in laccase activities of 180 and 43 U L−1 in the supernatant, respectively. The laccases, Sc-lac and Aa-lac, were applied to degrade lignin, achieving degradation rates of 26.92% and 11.54%, respectively. The optimal temperature and pH for both laccases were 60°C and pH 3–3.5. These findings provide valuable insights into community structure analysis via high-throughput sequencing and the laboratory selection of cultivable strains. Furthermore, the screening and application of these enzymes and microorganisms contribute to reducing environmental pollution and promoting a sustainable, eco-friendly environment.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143645694","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}

引用次数: 0

Pilot Scale Production of a F420 Precursor Under Microaerobic Conditions

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-03-18 DOI: 10.1002/biot.70002

Annika Lenić, Bettina Bardl, Florian Kloss, Gundela Peschel, Ivan Schlembach, Gerald Lackner, Lars Regestein, Miriam A. Rosenbaum

{"title":"Pilot Scale Production of a F420 Precursor Under Microaerobic Conditions","authors":"Annika Lenić, Bettina Bardl, Florian Kloss, Gundela Peschel, Ivan Schlembach, Gerald Lackner, Lars Regestein, Miriam A. Rosenbaum","doi":"10.1002/biot.70002","DOIUrl":"https://doi.org/10.1002/biot.70002","url":null,"abstract":"The functional investigation of redox cofactors is important for many potential biocatalytic processes, yet limited access to these molecules is often hampering efficient research activities. Deazaflavin-dependent enzymes mediate a range of biochemical redox reactions in prokaryotes. Coenzyme F420-dependent enzymes are interesting for asymmetric redox biocatalysis and other challenging reactions, but low F420 titers harvested from natural producers and engineered host strains so far limit intensive investigation of these enzymes. FO is a natural precursor of F420, which already shares many of the redox properties and was previously confirmed as a surrogate for F420 in certain enzymes. Here, we focused our research on the development of an overall process workflow from a 30-L pilot scale stirred tank bioprocess to an optimized downstream purification process to obtain pure FO from an engineered Escherichia coli host strain. We were able to shift the cofactor synthesis from riboflavin to FO via the implementation of oxygen-limited process conditions during heterologous fbiC expression and reached a final titer of 5.05 mg L−1 FO in our fermentation broth, which for the first time allows the purification of relevant amounts for subsequent experiments. Online fluorescence measurement within the bioreactor system made it possible to monitor FO formation and confirmed growth-associated FO biosynthesis.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143646173","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}

引用次数: 0