Biotechnology Journal最新文献_第6页

Development of Novel Annexin A5 Probes and a Fluorometric Microplate-Reader Method for High-Throughput Apoptosis Detection 用于细胞凋亡高通量检测的新型膜联蛋白A5探针和荧光微孔阅读器的开发

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70054

Mengyue Gao, Shihui Wang, Yunke Wang, Minjin Hu, Sheng Wang, Zichun Hua

{"title":"Development of Novel Annexin A5 Probes and a Fluorometric Microplate-Reader Method for High-Throughput Apoptosis Detection","authors":"Mengyue Gao, Shihui Wang, Yunke Wang, Minjin Hu, Sheng Wang, Zichun Hua","doi":"10.1002/biot.70054","DOIUrl":"https://doi.org/10.1002/biot.70054","url":null,"abstract":"<div>\u0000 \u0000 Annexin A5 (AnxA5) probes have been extensively utilized to investigate apoptotic cell death, which is crucial to various fields of biological research and clinical applications. Chemically labeled fluorescent AnxA5 has been widely used, but there is a notable scarcity of blue-emitting AnxA5 probes based on fluorescent proteins (FPs). Furthermore, methods utilizing AnxA5 predominantly rely on flow cytometry or fluorescence microscopy, both of which have inherent limitations, such as the necessity of costly instruments and specialized expertise, as well as the challenge of analyzing multiple samples. Fewer studies have focused on establishing a fluorometric microplate reader method for apoptosis detection. Herein, we reported a series of novel blue-emitting AnxA5 probes, ingeniously designed by fusing FPs. These probes can be applied for detecting apoptosis with flow cytometry and fluorescence microscopy. Additionally, based on our findings regarding the strong linearity between the fluorescence intensity and concentrations of AnxA5 probes, along with their analytical performances in apoptotic samples, we determined that blue-emitting AnxA5 probes were suitable for the microplate reader assay. We highlighted the reliability and practicality of this method by comparing with results obtained from flow cytometry. This fluorometric microplate reader assay offers a promising and high-throughput strategy for cancer researchers to evaluate the pro-apoptotic effects of potential antineoplastic drugs.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244404","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

Alleviating Substrate Inhibition of Leucine Dehydrogenase by Enhancing NAD+ Dissociation Efficiency 通过提高NAD+解离效率减轻底物对亮氨酸脱氢酶的抑制作用

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70045

Jun-Ping Zhou, Yi-Nan Xue, Feng Wang, Hui Gao, Zhi-Cheng Zhang, Ai-Ping Pang, Zhi-Qiang Liu, Yu-Guo Zheng

引用次数: 0

Development and Validation of a Three-Step Screening Strategy for Extracellular Salt-Tolerant Nucleases From Marine Bacteria 海洋细菌胞外耐盐核酸酶三步筛选策略的建立与验证

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70048

Aditya Achar, Subhojit Sen

{"title":"Development and Validation of a Three-Step Screening Strategy for Extracellular Salt-Tolerant Nucleases From Marine Bacteria","authors":"Aditya Achar, Subhojit Sen","doi":"10.1002/biot.70048","DOIUrl":"https://doi.org/10.1002/biot.70048","url":null,"abstract":"This study reports the development of a 3-step strategy that is both cost-effective and quick to screen marine organisms and validate the presence of extracellular nucleases. The assay plates (M9 or Luria Broth [LB] media with 500 mM salt) were overlaid with a thin layer of top agar containing Toluidine Blue as the indicator and salmon sperm DNA as the substrate. Primary screening of halophiles was based on their zone of clearance. Secondary screening of the isolates involved assaying the supernatants using a well-diffusion assay. The isolates were further screened and validated by ammonium sulfate fractionation of the cell-free supernatants to enrich the secreted nuclease. The three-step method narrowed down nine potential isolates from ∼500 bacterial colonies, of which SH1 demonstrated nuclease activity, discernibly due to a secreted extracellular enzyme(s). Further characterization of this enriched nuclease(s) showed that it is likely made up of multiple peptides/subunits, acts as an endo- as well as exonuclease, degrades both DNA and RNA, is Mg+2 dependent, has a wide range of salt tolerance from 80–1500 mM, is optimally active at 37°C, and is stable against reducing agents. This validates the screening strategy thus opening doors to further bioengineering of novel nucleases from other extremophiles.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70048","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244358","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

Replacement of Isoleucine and Leucine by their Keto Acids leads to increased formation of α-Hydroxy Acids in Chinese Hamster Ovary cells 异亮氨酸和亮氨酸被酮酸替代导致中国仓鼠卵巢细胞α-羟基酸的形成增加

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70041

Philipp Reifenberg, Lara Rosenberger, Maxime Le Mignon, Aline Zimmer

引用次数: 0

Recent Advances in Genome Base Editing Technology and Its Applications in Industrial Microorganism 基因组碱基编辑技术及其在工业微生物中的应用研究进展

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70052

Yi Zheng, Fei Du, Yiwen Hang, Wang Ma, Guang Yang, Na Wu, Xiaoman Sun

{"title":"Recent Advances in Genome Base Editing Technology and Its Applications in Industrial Microorganism","authors":"Yi Zheng, Fei Du, Yiwen Hang, Wang Ma, Guang Yang, Na Wu, Xiaoman Sun","doi":"10.1002/biot.70052","DOIUrl":"https://doi.org/10.1002/biot.70052","url":null,"abstract":"<div>\u0000 \u0000 Base editing technology is a novel gene-editing approach derived from the CRISPR/Cas9 system, enabling precise and efficient base conversion. Due to operational simplicity, strong target specificity, high editing efficiency, and minimal editing byproducts, base editing has been widely applied in gene therapy, crop breeding, construction of model organisms, and microbial metabolic engineering. In this review, we systematically summarize the development history and recent advancements of several major base editors, including cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR-free base editors, C-to-G base editors (CGBEs), glycosylase base editors (GBEs), and IscB-derived base editors. Furthermore, we comprehensively summarize optimization strategies for base editors and its application in constructing efficient industrial microorganism, such as Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus niger. This review aims to facilitate the broader application of base editing technologies in synthetic biology and accelerate their translational potential.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244354","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

A Two-Step Process for Converting Methane to Canthaxanthin Using Methylococcus capsulatus (Bath) Biomass and Engineered Yarrowia lipolytica 利用荚膜甲基球菌（Bath）生物质和工程解脂耶氏菌将甲烷转化为角黄素的两步法研究

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70043

Maria O. Taratynova, Ivan M. Tarasov, Iuliia M. Fedyaeva, Dmitry A. Dementev, Veronika A. Gorchakova, Margarita A. Tarasova, Alexander S. Fedorov, Tigran V. Yuzbashev, Sergey P. Sineoky, Evgeniya Y. Yuzbasheva

引用次数: 0

Simultaneous Repression of GLUCAN WATER DIKINASE 1 and STARCH BRANCHING ENZYME 1 in Potato Tubers Leads to Starch With Increased Amylose and Novel Industrial Properties 马铃薯块茎中葡聚糖水二激酶1和淀粉分支酶1的同时抑制导致淀粉直链淀粉增加和新的工业特性

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70051

Muyiwa S. Adegbaju, Nina Gouws, Christell van der Vyver, Pedri Claassens, Jens Kossmann, Michaela Fischer-Stettler, Samuel C. Zeeman, James R. Lloyd

{"title":"Simultaneous Repression of GLUCAN WATER DIKINASE 1 and STARCH BRANCHING ENZYME 1 in Potato Tubers Leads to Starch With Increased Amylose and Novel Industrial Properties","authors":"Muyiwa S. Adegbaju, Nina Gouws, Christell van der Vyver, Pedri Claassens, Jens Kossmann, Michaela Fischer-Stettler, Samuel C. Zeeman, James R. Lloyd","doi":"10.1002/biot.70051","DOIUrl":"https://doi.org/10.1002/biot.70051","url":null,"abstract":"This study examines how post-transcriptional gene silencing of STARCH BRANCHING ENZYME 1 (SBE1) and GLUCAN WATER DIKINASE 1 (GWD1) affects the structure and properties of potato tuber starch. Silencing of either gene individually or simultaneously altered starch chemistry physical properties. Repression of StGWD1 reduced phosphate content, while repression of StSBE1 increased it. The phosphate content of starch isolated from plants where both genes were repressed was increased compared to StGWD1 repressed lines, but lower than both the SBE1 repressed lines and the untransformed control. Constituent chain lengths of starches from all lines were altered, and amylose content was increased in the gwd1 and sbe1/gwd1 double repressed lines, which also accumulated small numbers of lobed starch granules. Pasting properties were also affected, with starch from StSBE1-repressed lines demonstrating increased peak and trough viscosities and gwd1 lines showing decreased peak and trough viscosities, compared with the control. Peak and trough viscosities were lowest in the sbe1/gwd1 repressed lines. We believe that these data demonstrate that alterations in starch phosphate influence the degree of branching within starch and offer a novel in planta strategy for optimizing the industrial properties of potato storage starch.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244232","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

Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System 通过T7 RNAP/CRISPRa系统激活法菲Komagataella的合成遗传电路

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70036

Shupeng Ruan, Aoxue Wang, Hongyi Zou, Ying Lin, Lei Ye, Shuli Liang

{"title":"Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System","authors":"Shupeng Ruan, Aoxue Wang, Hongyi Zou, Ying Lin, Lei Ye, Shuli Liang","doi":"10.1002/biot.70036","DOIUrl":"https://doi.org/10.1002/biot.70036","url":null,"abstract":"<div>\u0000 \u0000 The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine-tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7-tRNA-sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet-on induction system, split-T7 system, and RNA cleavage processing by HH-HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self-responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244237","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

Engineering Saccharomyces Cerevisiae With Novel Functional Xylose Isomerases From Rumen Microbiota for Enhanced Biofuel Production 利用瘤胃微生物群中新型功能木糖异构酶对酿酒酵母进行工程改造，以提高生物燃料产量

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-06-09 DOI: 10.1002/biot.70050

Beatriz de Oliveira Vargas, Marcelo Falsarella Carazzolle, Juliana Pimentel Galhardo, Juliana José, Brenda Cristina de Souza, Jéssica Batista de Lima Correia, Jade Ribeiro dos Santos, Gonçalo Amarante Guimarães Pereira, Fellipe da Silveira Bezerra de de Mello

{"title":"Engineering Saccharomyces Cerevisiae With Novel Functional Xylose Isomerases From Rumen Microbiota for Enhanced Biofuel Production","authors":"Beatriz de Oliveira Vargas, Marcelo Falsarella Carazzolle, Juliana Pimentel Galhardo, Juliana José, Brenda Cristina de Souza, Jéssica Batista de Lima Correia, Jade Ribeiro dos Santos, Gonçalo Amarante Guimarães Pereira, Fellipe da Silveira Bezerra de de Mello","doi":"10.1002/biot.70050","DOIUrl":"https://doi.org/10.1002/biot.70050","url":null,"abstract":"Xylose metabolism in Saccharomyces cerevisiae remains a significant bottleneck due to the difficulty in identifying functional and efficient xylose isomerases (XI). In the present study, publicly available metagenomic and metatranscriptomic datasets of rumen microbiota from different herbivorous mammals were used to prospect novel XIs sequences. Seven putative XIs from moose, camel, cow, and sheep were cloned into a strain modified for xylose metabolism. Out of those, five XIs demonstrated activity and efficiently converted xylose into xylulose, resulting in ethanol as the final product. A XI from camel rumen microbiota exhibited a KM of 16.25 mM, indicating high substrate affinity. The strains expressing enzymes XI11 and XI12, obtained from sheep rumen microbiota, were able to deplete 40 g/L of xylose within 72 and 96 h, achieving theoretical ethanol yields of 90% and 88%, respectively. These results are comparable to those obtained with Orpinomyces sp. ukk1 XI, a benchmark enzyme previously reported as highly efficient in S. cerevisiae. This study also provides the first report on the successful expression of XIs mined from the ruminal microbiotas of sheep and camels in S. cerevisiae, expanding the perspectives for the optimization of fermentation processes and the production of lignocellulosic biofuels from xylose.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70050","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244233","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

Arg128*-Mediated Dual-Substrate Recognition and Dynamic Transport Mechanisms in (R)-ω-Transaminase: Computational Insights and Mutational Profiling Guided Rational Engineering (R) ω-转氨酶中Arg128*介导的双底物识别和动态转运机制：计算见解和突变谱指导的理性工程

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2025-05-19 DOI: 10.1002/biot.70032

Jie Chen, Shuai Qiu, Conglin Ju, Dan Wang, Fangfang Fan, Jun Huang

{"title":"Arg128*-Mediated Dual-Substrate Recognition and Dynamic Transport Mechanisms in (R)-ω-Transaminase: Computational Insights and Mutational Profiling Guided Rational Engineering","authors":"Jie Chen, Shuai Qiu, Conglin Ju, Dan Wang, Fangfang Fan, Jun Huang","doi":"10.1002/biot.70032","DOIUrl":"https://doi.org/10.1002/biot.70032","url":null,"abstract":"<div>\u0000 \u0000 ω-Transaminases (ω-TAs) are critical biocatalysts for the asymmetric synthesis of chiral amines, and uniquely accommodate both hydrophobic and hydrophilic substrates through a conserved binding pocket. In this study, we combine computational simulations and site-directed mutagenesis to dissect this dual-function structure of (R)-selective ω-transaminase from Aspergillus terreus (AtATA). Our results reveal that AtATA employs a synergistic mechanism: aromatic residues within the large pocket stabilize hydrophobic substrates via π-driven interactions, while Arg128* dynamically interacts with hydrophilic compounds through hydrogen bonding. Furthermore, the binding pocket of AtATA exhibits remarkable plasticity to accommodate diverse substrates, with the side chain of Arg128* dynamically adjusting its conformation to facilitate the transport of substrates. Mutational profiling, particularly the R128*A mutation, directly validates these mechanistic insights. Our finding reveals the Arg128*-mediated dual-substrate recognition and transport mechanisms, providing a solid theoretical foundation for enhancing the industrial application of transaminases in pharmaceutical synthesis and green chemistry.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144091271","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