Journal of biotechnology最新文献

Metabolic engineering of Cupriavidus necator using sucrose phosphorylase pathway for polyhydroxybutyrate production from sucrose. 蔗糖磷酸化酶途径下赤铜产聚羟基丁酸盐的代谢工程研究。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-11-01 Epub Date: 2025-08-05 DOI: 10.1016/j.jbiotec.2025.08.002

Vijaykumar Khonde, Mandar Deshpande, Dheeraj Mahajan, Meenakshi Tellis, Pramod Kumbhar, Anand Ghosalkar

{"title":"Metabolic engineering of Cupriavidus necator using sucrose phosphorylase pathway for polyhydroxybutyrate production from sucrose.","authors":"Vijaykumar Khonde, Mandar Deshpande, Dheeraj Mahajan, Meenakshi Tellis, Pramod Kumbhar, Anand Ghosalkar","doi":"10.1016/j.jbiotec.2025.08.002","DOIUrl":"10.1016/j.jbiotec.2025.08.002","url":null,"abstract":"<p><p>Sucrose-rich feedstocks are the most suitable raw materials for the production of biodegradable polymers like Polyhydroxyalkanoates (PHAs). Cupriavidus necator, a versatile microorganism with natural ability to accumulate poly(3-hydroxybutyrate) (PHB), has been shown to utilize a diverse set of carbon sources including sugars, oils, and gaseous feedstock like CO<sub>2</sub>. However, both wild-type and mutant strains of C. necator cannot metabolize sucrose, limiting its utility in industrial production using sucrose-rich feedstocks. We developed metabolically engineered strains of C. necator for sucrose utilization by introducing sucrose phosphorylase pathway. Among all the recombinant strains, C. necator harbouring sucrose phosphorylase from Rhizobium vitis (CN-SPrv) along with sucrose permease and phosphoglucomutase from Escherichia coli demonstrated the most efficient utilization of sucrose. The CN-SPrv strain was evaluated for the utilization of sucrose using cane biosyrup and resulted in 60 g/L of PHB titer and 31 % yield on a consumed sugar basis in fed-batch mode of fermentation. This is the first report on metabolic engineering of C. necator using the sucrose phosphorylase pathway for PHB production.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"22-30"},"PeriodicalIF":3.9,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144794555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Modular engineering of the doxorubicin biosynthetic gene cluster based on component functional clustering in Streptomyces. 基于链霉菌组分功能聚类的多柔比星生物合成基因簇模块化工程

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-10-03 DOI: 10.1016/j.jbiotec.2025.09.019

Tianqi Cui, Chanjuan Jiang, Jiale Li, Shuo Wang, Lingdi Li, Ji Luan, Hailong Wang

{"title":"Modular engineering of the doxorubicin biosynthetic gene cluster based on component functional clustering in Streptomyces.","authors":"Tianqi Cui, Chanjuan Jiang, Jiale Li, Shuo Wang, Lingdi Li, Ji Luan, Hailong Wang","doi":"10.1016/j.jbiotec.2025.09.019","DOIUrl":"10.1016/j.jbiotec.2025.09.019","url":null,"abstract":"<p><p>The anticancer drug doxorubicin is synthesized through the synergistic action of the type II polyketide synthases, along with an array of modification enzymes in Streptomyces. The 33 doxorubicin biosynthetic genes are scattered across at least 15 distinct transcriptional units, posing challenges for their regulatory control. In this study, we firstly achieved heterologous production of doxorubicin in a range of Streptomyces hosts by supplementing functional genes essential for biosynthesis of the sugar moiety. Then, employed a modular engineering approach to reconstruct the doxorubicin gene cluster, grouping the genes responsible for each biosynthetic module into 6 well-defined subclusters. Based on these 6 subclusters, we identified that the glycosylation and post-modification subcluster is the module with the greatest capacity to boost doxorubicin production. When introduced into the heterologous host S. albus J1074, the combination of the reconstructed modular gene cluster and the glycosylation and post-modification module resulted in a doxorubicin production rate that was 15 times greater than that of the natural gene cluster. Modular engineering of the doxorubicin gene cluster, which involves clustering based on component functionality, will streamline the regulatory control of the biosynthetic pathway and facilitate its transplantation into desired hosts for production optimization.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"265-274"},"PeriodicalIF":3.9,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Enhancement of natamycin production in Streptomyces gilvosporeus through heavy ion beam mutagenesis and global transcription machinery engineering. 通过重离子束诱变和全局转录机械工程提高gilvosporeus链霉菌纳他霉素的产量。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-10-03 DOI: 10.1016/j.jbiotec.2025.09.017

Yuxiu Xu, Liang Wang, Wei Hu, Jian Xue, Wen Xiao, Hongjian Zhang, Jianhua Zhang, Xusheng Chen

{"title":"Enhancement of natamycin production in Streptomyces gilvosporeus through heavy ion beam mutagenesis and global transcription machinery engineering.","authors":"Yuxiu Xu, Liang Wang, Wei Hu, Jian Xue, Wen Xiao, Hongjian Zhang, Jianhua Zhang, Xusheng Chen","doi":"10.1016/j.jbiotec.2025.09.017","DOIUrl":"10.1016/j.jbiotec.2025.09.017","url":null,"abstract":"<p><p>Natamycin is an antifungal agent produced by Streptomyces and is widely used in food, pharmaceuticals, and other fields. However, the low production of natamycin limits its application in various fields, making it crucial to enhance the biosynthetic capacity of natamycin-producing strains. In this study, Streptomyces gilvosporeus ATCC 13326 was used to construct a chassis for the efficient production of natamycin through strain engineering. A mutant strain S. gilvosporeus Z1403 with a titer of 1.7 g·L<sup>-1</sup> was obtained by heavy ion beam radiation and LiCl resistance screening, 70.0 % higher than that of the wild-type strain S. gilvosporeus ATCC 13326. Subsequently, transcription factor screening identified whiG as a key regulator of natamycin biosynthesis. Targeting whiG via global transcription machinery engineering further enhanced natamycin production of S. gilvosporeus Z1403. Finally, the high-production mutant strain EP-whiG was obtained with a titer of 2.2 g·L<sup>-1</sup>, 29.4 % and 120.0 % higher than that of the parent strain S. gilvosporeus Z1403 and the wild-type strain, respectively. S. gilvosporeus EP-whiG achieved a natamycin production of 13.1 g·L<sup>-1</sup> in a 5-L bioreactor within 120 h by fed-batch fermentation, which was 61.7 % higher than that of the wild-type strain. These results suggest that combining heavy ion beam mutagenesis with global transcription machinery engineering is an effective strategy for strain improvement, laying a theoretical foundation for enhancing the production of secondary metabolites in Streptomyces.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"254-264"},"PeriodicalIF":3.9,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232628","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Optimizing Chlorella vulgaris production and exploring its impact on germination through microalga-N₂-fixing bacteria consortia. 优化小球藻产量及探讨微藻-固氮菌群对其萌发的影响。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-10-01 DOI: 10.1016/j.jbiotec.2025.09.018

Ana Sanchez-Zurano, Silvia Vilaró-Cos, Daniel Figueiredo, Lusine Melkonyan, Alice Ferreira, Francisco Gabriel Acién, Tomas Lafarga, Luisa Gouveia

{"title":"Optimizing Chlorella vulgaris production and exploring its impact on germination through microalga-N<sub>2</sub>-fixing bacteria consortia.","authors":"Ana Sanchez-Zurano, Silvia Vilaró-Cos, Daniel Figueiredo, Lusine Melkonyan, Alice Ferreira, Francisco Gabriel Acién, Tomas Lafarga, Luisa Gouveia","doi":"10.1016/j.jbiotec.2025.09.018","DOIUrl":"10.1016/j.jbiotec.2025.09.018","url":null,"abstract":"<p><p>Microalgal biomass is increasingly valued in industrial and agricultural sectors due to its bioactive compounds. However, large-scale production remains costly, mainly due to nitrogen fertilizer expenses. A promising sustainable alternative is co-cultivation with N<sub>2</sub>-fixing bacteria, capable of supplying biologically available nitrogen. In this study, Chlorella vulgaris was grown in synthetic medium with and without nitrogen, as well as in co-culture with three different N<sub>2</sub>-fixing bacteria in nitrogen-free medium. Microalgal growth was assessed by dry weight, Fv/Fm ratio, and flow cytometry, which also allowed evaluation of population dynamics and cell viability. Biomass composition (proteins, carbohydrates, lipids, chlorophyll, and carotenoids) was analyzed under all conditions. Co-cultures in nitrogen-free medium showed comparable biomass productivity to nitrogen-supplemented controls, although Fv/Fm values indicated physiological stress in some cases. Moreover, the agricultural potential of the resulting biomass and supernatants was evaluated through germination bioassays using lettuce seeds. All cultures tested at 0.2 g·L<sup>-1</sup> significantly improved the germination index. Also, applying the culture supernatant (biomass removed) also yielded positive effects, with GI increases exceeding 40 %. These results suggest that co-cultivation with N<sub>2</sub>-fixing bacteria can support efficient microalgal production while generating biomass and supernatants with biostimulant potential, contributing to sustainable agriculture and circular bioeconomy strategies.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"244-253"},"PeriodicalIF":3.9,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145225246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Komagataella phaffii as a microbial cell factory for antimicrobial peptide production. Komagataella phaffii作为抗菌肽生产的微生物细胞工厂。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-30 DOI: 10.1016/j.jbiotec.2025.09.015

Michel Lopes Leite, Kamila Botelho Sampaio de Oliveira, Letícia Ferreira Lima, Nadielle Tamires Moreira Melo, José Brango-Vanegas, Hugo Costa Paes, Octávio Luiz Franco

{"title":"Komagataella phaffii as a microbial cell factory for antimicrobial peptide production.","authors":"Michel Lopes Leite, Kamila Botelho Sampaio de Oliveira, Letícia Ferreira Lima, Nadielle Tamires Moreira Melo, José Brango-Vanegas, Hugo Costa Paes, Octávio Luiz Franco","doi":"10.1016/j.jbiotec.2025.09.015","DOIUrl":"10.1016/j.jbiotec.2025.09.015","url":null,"abstract":"<p><p>In recent decades, the methylotrophic yeast Komagataella phaffii has emerged as a powerful host for the heterologous production of antimicrobial peptides (AMPs) as an economical and scalable platform. K. phaffii combines several important advantages for a recombinant expression system, such as rapid growth, high-density cell culture, efficient protein secretion, and the ability to perform essential post-translational modifications. The methanol-inducible AOX1 promoter (P<sub>AOX1</sub>), generally employed through the pPICZalpha vector, allows strong and tightly regulated heterologous expression and is a central factor in the expression system of this yeast. However, several strategies have been employed to produce recombinant AMPs in K. phaffii, among them codon optimization, engineered methanol-resistant strains, alternative promoters, and the use of different secretory peptides. This review highlights the latest advances and practical considerations in the use of K. phaffii for AMP production, discussing challenges such as peptide stability, proteolytic degradation, and yield optimization. The insights provided contribute to the expansion of biotechnological applications of K. phaffii, reinforcing its potential as an efficient and safe platform for large-scale production of therapeutic AMPs.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"275-287"},"PeriodicalIF":3.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145212745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

High-concentration Mg2+ stress improves L-lactic acid biosynthesis of Bacillus coagulans revealed by combined analysis of transcriptome and metabolome 转录组和代谢组联合分析表明高浓度Mg2+胁迫促进了凝结芽孢杆菌l -乳酸的生物合成。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-30 DOI: 10.1016/j.jbiotec.2025.09.016

Zhitong Sun , Jiaxin Liang , Gang Wang , Yumeng Zhen , Jinlong Liu , Di Cai , Bin Wang , Yong Wang

{"title":"High-concentration Mg2+ stress improves L-lactic acid biosynthesis of Bacillus coagulans revealed by combined analysis of transcriptome and metabolome","authors":"Zhitong Sun , Jiaxin Liang , Gang Wang , Yumeng Zhen , Jinlong Liu , Di Cai , Bin Wang , Yong Wang","doi":"10.1016/j.jbiotec.2025.09.016","DOIUrl":"10.1016/j.jbiotec.2025.09.016","url":null,"abstract":"<div><div>The application of magnesium oxide as a neutralizing agent significantly enhanced L-lactic acid (L-LA) production in <em>Bacillus coagulans</em> (<em>B. coagulans</em>), increasing titer, yield, productivity, and cell viability by 21.81 %, 7.61 %, 22.22 %, and 18.50 times, respectively. To elucidate the metabolic response to high-concentration Mg<sup>2+</sup> stress, transcriptomic analysis identified 1021 differentially expressed genes (DEGs), with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealing predominant biological processes and pathway dysregulation. Metabolomic profiling detected 1196 differential metabolites, of which 216 were annotated to 129 metabolic pathways. Integrated multi-omics analyses delineated 10 core pathways involving 139 DEGs and 29 metabolites, demonstrating that Mg<sup>2+</sup> stress enhances lactic acid fermentation efficiency through reprogramming of carbon flux, nucleic acid biosynthesis, amino acid metabolism, cofactor dynamics, membrane transport, and transcriptional regulation. This study provides mechanistic insights into <em>B. coagulans</em> adaptation to Mg<sup>2+</sup> stress and proposes a viable strategy to optimize industrial L-LA bioproduction.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 1-13"},"PeriodicalIF":3.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145212742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Chitosan application in cosmetics and dermatology – Antimicrobial and prebiotic potential to control human microbiome 壳聚糖在化妆品和皮肤病学中的应用-抗菌和益生元控制人体微生物群的潜力。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-27 DOI: 10.1016/j.jbiotec.2025.09.014

Masha Delic, Irina Butorova, Andrey Kuskov

{"title":"Chitosan application in cosmetics and dermatology – Antimicrobial and prebiotic potential to control human microbiome","authors":"Masha Delic, Irina Butorova, Andrey Kuskov","doi":"10.1016/j.jbiotec.2025.09.014","DOIUrl":"10.1016/j.jbiotec.2025.09.014","url":null,"abstract":"<div><div>The human microbiome has become a critically important field of study, with recent advances continuously revealing new aspects of its functional roles. A deeper understanding of the complex metabolic interactions between the microbiome, therapeutic agents, and the human host is now developing. A key challenge is the development of effective novel antimicrobial compounds, particularly those targeting biofilm-related infections implicated in a wide range of chronic diseases, to better control the human microbiota and treat typical skin diseases. Chitosan, a polysaccharide derived from various natural sources, possesses a unique set of biological properties, making it a promising base for numerous cosmetic and medicinal products aimed at managing dermatological diseases and modulating the microbiome. By altering its characteristics, chitosan's properties can be tuned to produce biomacromolecules with optimized activity beneficial to human microbiota. This review summarizes the sources and mechanisms underlying chitosan's antimicrobial activity, its immunomodulatory and anti-pathogenic effects, and critically evaluates its dual potential as both an antimicrobial and a prebiotic agent. Special emphasis is placed on chitosan-based formulations for treating skin diseases and promoting microbiome health – an emerging and promising area of research.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 217-231"},"PeriodicalIF":3.9,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145191686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Seaweed-derived biostimulants for sustainable crop production: A review 海藻衍生生物刺激素用于可持续作物生产：综述。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-27 DOI: 10.1016/j.jbiotec.2025.09.013

Mohamed Lamine Rabhi , Larbi Derbak , Hamdi Bendif , Fehmi Boufahja , Abdelghafar Mohamed Abu-Elsaoud , Stefania Garzoli

{"title":"Seaweed-derived biostimulants for sustainable crop production: A review","authors":"Mohamed Lamine Rabhi , Larbi Derbak , Hamdi Bendif , Fehmi Boufahja , Abdelghafar Mohamed Abu-Elsaoud , Stefania Garzoli","doi":"10.1016/j.jbiotec.2025.09.013","DOIUrl":"10.1016/j.jbiotec.2025.09.013","url":null,"abstract":"<div><div>The increasing concerns over climate changes and environmental impacts of synthetic agrochemicals have led to a growing interest in natural alternatives such as seaweed-based biostimulants. These extracts contain diverse bioactive compounds, including phytohormones, polysaccharides, proteins, polyphenols, and vitamins, which contribute to enhanced plant growth, stress tolerance, and soil health. Seaweed biostimulants have demonstrated their ability to improve germination, growth, and yield under both optimal and stressful conditions. Additionally, seaweed-derived compounds influence microbial communities, promoting beneficial interactions that enhance soil structure and fertility. Their different mode of applications further expands their utility in modern agriculture. Despite their numerous advantages, challenges remain regarding cultivation, large-scale production, and regulatory frameworks. Further research is needed to optimize extraction methods, elucidate mechanisms of action, and explore economic feasibility. This systematic review brings focus to the potential of seaweed-based biostimulants as sustainable agricultural inputs, discussing their chemical composition, mechanisms of action, techniques of extraction, application strategies, challenges and future perspectives for improving crop productivity and resilience.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 201-216"},"PeriodicalIF":3.9,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145191693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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 next-generation platform for highly optimized CRISPR-mediated transcriptional repression 高度优化crispr介导的转录抑制的下一代平台。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-24 DOI: 10.1016/j.jbiotec.2025.09.012

Andrew Kristof , Krithika Karunakaran , Yann Ferry , Paula Mizote , Christopher Allen , Sophie Briggs , John Blazeck

{"title":"A next-generation platform for highly optimized CRISPR-mediated transcriptional repression","authors":"Andrew Kristof , Krithika Karunakaran , Yann Ferry , Paula Mizote , Christopher Allen , Sophie Briggs , John Blazeck","doi":"10.1016/j.jbiotec.2025.09.012","DOIUrl":"10.1016/j.jbiotec.2025.09.012","url":null,"abstract":"<div><div>CRISPR interference (CRISPRi), the fusion of transcriptional repressor domains to nuclease-inactive Cas9, is a powerful genetic tool enabling site-specific suppression of gene expression. However, its performance remains inconsistent across cell lines, gene targets, or single guide RNAs (sgRNAs) employed. This study described the development process of a novel, highly optimized CRISPRi repressor for mammalian gene regulation through a multi-pronged protein engineering approach: (1) truncating established domains, (2) characterizing candidate domains, (3) creating combinatorial domain fusion libraries, and (4) optimizing NLS configuration. First, by evaluating several truncations of MeCP2, a well-established repressor, we see that the ultra-compact NCoR/SMRT interaction domain (NID) significantly enhances CRISPRi gene knockdown performance, exceeding levels observed with canonical MeCP2 subdomains by an average of ∼40 %. Incorporating this optimized MeCP2 NID truncation with a diverse panel of authenticated repressor domains, we next assemble and screen combinatorial multi-domain libraries, discovering four new repressor fusions. Upon follow-up nuclear localization signal (NLS) configuration analysis, we see that affixing one carboxy-terminal NLS enhances gene knockdown efficiency of the repressors by an average of ∼50 %. Through rigorous validation of NLS-tagged repressor fusions across several cell lines, multiple sgRNA targets, and genome-wide dropout screens, we establish that our strongest system, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing capabilities over alternative CRISPRi platforms. In addition to developing dCas9-ZIM3-NID-MXD1-NLS, a uniquely potent transcriptional repressor, we envision that the multi-domain engineering approach utilized in this study will be valuable framework enabling future strides in CRISPR platform development.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 192-200"},"PeriodicalIF":3.9,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145175621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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

Improving thermostability of α-L-fucosidase from Pedobacter sp. via consensus-guided engineering and directed evolution 通过共识导向工程和定向进化提高Pedobacter sp. α-L- focusidase的热稳定性。

IF 3.9 2区生物学

Journal of biotechnology Pub Date : 2025-09-22 DOI: 10.1016/j.jbiotec.2025.09.011

Tong-Tong Zhao , Yu-Bo Wang , Wen-Qian Qiu , Ying-Ying Wang , Yang-Yong Lv , Huan-Chen Zhai , Yuan-Sen Hu , Zheng-Qiang Jiang , Shuai-Bing Zhang

{"title":"Improving thermostability of α-L-fucosidase from Pedobacter sp. via consensus-guided engineering and directed evolution","authors":"Tong-Tong Zhao , Yu-Bo Wang , Wen-Qian Qiu , Ying-Ying Wang , Yang-Yong Lv , Huan-Chen Zhai , Yuan-Sen Hu , Zheng-Qiang Jiang , Shuai-Bing Zhang","doi":"10.1016/j.jbiotec.2025.09.011","DOIUrl":"10.1016/j.jbiotec.2025.09.011","url":null,"abstract":"<div><div>The α-L-fucosidase from <em>Pedobacter</em> sp. (<em>Pb</em>Fuc), a glycoside hydrolase capable of catalyzing the synthesis of 2’-fucosyllactose (2’-FL) from 4-nitrophenyl α-L-fucopyranoside (<em>p</em>NP-Fuc) and D-lactose, exhibits limited industrial applicability due to inherent thermostability constraints. This study implemented a combinatorial strategy integrating consensus-guided engineering and directed evolution to engineer the thermal stability of <em>Pb</em>Fuc, resulting in the identification of six critical mutants (G53C, M54I, N59S, T71H, R125C, S199P) and the subsequent construction of the combinatorial mutant M6. Thermostability assays revealed complete inactivation of the wild-type enzyme after 30-min incubation at 45 °C, whereas M6 retained approximately 40 % residual activity under equivalent conditions at 60 °C, accompanied by an increase in the optimal reaction temperature from 35 °C to 40 °C. Structural mechanism analysis demonstrated that the enhanced thermostability of M6 originated from synergistic multilevel structural optimization and reorganization of molecular interaction networks: Conformational stabilization manifested through prolonged maintenance of stable secondary structural conformations during thermal stress and reduced amplitude of tertiary structural fluctuations; Global structural compaction decreased solvent-accessible surface area, thereby minimizing thermal energy transfer; Local structural reinforcement occurred via the formation of novel hydrogen bonds, enhanced rigidity through π-π stacking, and neutralization of electrostatic repulsion via charge compensation.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 168-180"},"PeriodicalIF":3.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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