Pub Date : 2025-09-30DOI: 10.1016/j.jbiotec.2025.09.016
Zhitong Sun , Jiaxin Liang , Gang Wang , Yumeng Zhen , Jinlong Liu , Di Cai , Bin Wang , Yong Wang
The application of magnesium oxide as a neutralizing agent significantly enhanced L-lactic acid (L-LA) production in Bacillus coagulans (B. coagulans), 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 Mg2+ 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 Mg2+ 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 B. coagulans adaptation to Mg2+ stress and proposes a viable strategy to optimize industrial L-LA bioproduction.
{"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}
Pub Date : 2025-09-27DOI: 10.1016/j.jbiotec.2025.09.014
Masha Delic, Irina Butorova, Andrey Kuskov
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.
{"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}
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.
{"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}
Pub Date : 2025-09-24DOI: 10.1016/j.jbiotec.2025.09.012
Andrew Kristof , Krithika Karunakaran , Yann Ferry , Paula Mizote , Christopher Allen , Sophie Briggs , John Blazeck
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.
{"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}
Pub Date : 2025-09-22DOI: 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
The α-L-fucosidase from Pedobacter sp. (PbFuc), a glycoside hydrolase capable of catalyzing the synthesis of 2’-fucosyllactose (2’-FL) from 4-nitrophenyl α-L-fucopyranoside (pNP-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 PbFuc, 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.
{"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}
Pub Date : 2025-09-22DOI: 10.1016/j.jbiotec.2025.09.010
Qingyuan Ran , Xinran Zhang , Chen Wang , Weijian Zhang , Liang Zhao , Wen-Song Tan , Qian Ye
Hyperosmotic stimulation is a prevalent strategy to enhance cell culture productivity in fed-batch cultures. Maintaining stable hyperosmotic conditions during perfusion cultures presents a promising strategy, but research on its application in perfusion processes remains limited. In this study, we investigated cellular responses under constant hyperosmolality in Chinese hamster ovary (CHO) cell perfusion cultures using Raman spectroscopy to maintain a constant hyperosmotic environment. Integrated genome-scale metabolic model (GEM) with real-time monitoring of the oxygen uptake rate (OUR) was employed to systematically analyze the metabolic alterations induced by constant hyperosmotic stimulation. Our findings showed that the CHO cells exhibited time-dependent metabolic responses, with rapid changes in nutrient uptake, glycolysis, and TCA cycle activity, while lactate metabolism responded more slowly. The specific productivity (qmAb) displayed the slowest changes, stabilizing only after 6–9 days upon the simulation, resulting in a maximum increase up to 168.5 %. Notably, we found shifts in the intracellular redox environment, and the cells enhanced their antioxidative capacity at low dissolved oxygen (DO) levels under hyperosmotic conditions. Even when DO dropped to 10 %, the cells subjected to hyperosmotic stimulation maintained relatively low levels of reactive oxygen species (ROS) while preserving high qmAb. Overall, this study provides new insights into cellular responses under constant hyperosmotic condition and provides insights for the application of hyperosmotic strategies in perfusion processes.
{"title":"Genome-scale metabolic analysis reveals enhanced metabolism and antioxidative stress response in perfusion cell culture under constant hyperosmotic stimulation","authors":"Qingyuan Ran , Xinran Zhang , Chen Wang , Weijian Zhang , Liang Zhao , Wen-Song Tan , Qian Ye","doi":"10.1016/j.jbiotec.2025.09.010","DOIUrl":"10.1016/j.jbiotec.2025.09.010","url":null,"abstract":"<div><div>Hyperosmotic stimulation is a prevalent strategy to enhance cell culture productivity in fed-batch cultures. Maintaining stable hyperosmotic conditions during perfusion cultures presents a promising strategy, but research on its application in perfusion processes remains limited. In this study, we investigated cellular responses under constant hyperosmolality in Chinese hamster ovary (CHO) cell perfusion cultures using Raman spectroscopy to maintain a constant hyperosmotic environment. Integrated genome-scale metabolic model (GEM) with real-time monitoring of the oxygen uptake rate (OUR) was employed to systematically analyze the metabolic alterations induced by constant hyperosmotic stimulation. Our findings showed that the CHO cells exhibited time-dependent metabolic responses, with rapid changes in nutrient uptake, glycolysis, and TCA cycle activity, while lactate metabolism responded more slowly. The specific productivity (<em>q</em><sub><em>mAb</em></sub>) displayed the slowest changes, stabilizing only after 6–9 days upon the simulation, resulting in a maximum increase up to 168.5 %. Notably, we found shifts in the intracellular redox environment, and the cells enhanced their antioxidative capacity at low dissolved oxygen (DO) levels under hyperosmotic conditions. Even when DO dropped to 10 %, the cells subjected to hyperosmotic stimulation maintained relatively low levels of reactive oxygen species (ROS) while preserving high <em>q</em><sub><em>mAb</em></sub>. Overall, this study provides new insights into cellular responses under constant hyperosmotic condition and provides insights for the application of hyperosmotic strategies in perfusion processes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 181-191"},"PeriodicalIF":3.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137658","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}
Pub Date : 2025-09-19DOI: 10.1016/j.jbiotec.2025.09.009
Ling Zou , Jian Liu , Yidan Hu , Yan Zhao , Xiaobo Liu
Microbial melanin production traditionally relies on expensive tyrosine supplementation, making the cost-effective production of melanin challenging. To address this substrate dependency, we engineered an Escherichia coli strain capable of autonomous tyrosine synthesis through the co-overexpression of the tyrosine synthetase gene (aroGfbr or tyrAfbr with feedback resistance) and the tyrosinase gene tyr1. By further integrating the tyrosinase gene tyr1 into this engineered strain, we achieved melanin yields ranging from 5.58 to 7.57 mg/mL in the absence of exogenous tyrosine, thereby reducing production costs by 70 %–73.33 % compared to conventional methods that require exogenous tyrosine supplementation. This study establishes a robust and cost-effective platform for sustainable melanin production, with significant implications for the industrial manufacturing of microbial pigments.
{"title":"Cost-effective melanin production using engineered Escherichia coli with autonomous tyrosine biosynthesis","authors":"Ling Zou , Jian Liu , Yidan Hu , Yan Zhao , Xiaobo Liu","doi":"10.1016/j.jbiotec.2025.09.009","DOIUrl":"10.1016/j.jbiotec.2025.09.009","url":null,"abstract":"<div><div>Microbial melanin production traditionally relies on expensive tyrosine supplementation, making the cost-effective production of melanin challenging. To address this substrate dependency, we engineered an <em>Escherichia coli</em> strain capable of autonomous tyrosine synthesis through the co-overexpression of the tyrosine synthetase gene (<em>aroG</em><sup><em>fbr</em></sup> or <em>tyrA</em><sup><em>fbr</em></sup> with feedback resistance) and the tyrosinase gene <em>tyr1</em>. By further integrating the tyrosinase gene <em>tyr1</em> into this engineered strain, we achieved melanin yields ranging from 5.58 to 7.57 mg/mL in the absence of exogenous tyrosine, thereby reducing production costs by 70 %–73.33 % compared to conventional methods that require exogenous tyrosine supplementation. This study establishes a robust and cost-effective platform for sustainable melanin production, with significant implications for the industrial manufacturing of microbial pigments.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 151-157"},"PeriodicalIF":3.9,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145096192","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}
Pub Date : 2025-09-18DOI: 10.1016/j.jbiotec.2025.09.008
Elizabeth Matthews , Neha Tushar Dalvi , Michael Butler
Glycan profiles of monoclonal antibodies are critical quality attributes for their use as therapeutic agents. However, the control of glycosylation can prove difficult during large-scale manufacture. An on-going problem is the increase in high-mannose glycans (HM) that can occur unexpectedly under cell culture conditions that disrupt the glycosylation pathway. We have developed a method for removal of high-mannose glycans from a heterogeneous mixture of mAb glycoforms purified from a bioprocess. This involves the use of a prokaryotic lectin (RPL-Man2) bound to agarose beads to selectively bind reversibly to mAbs carrying HM-glycans. The unbound flow-through from this step contains the remainder of the mAb preparation with a reduced HM-glycan content. In this paper we demonstrate the application of this method using two distinct antibodies derived from Chinese hamster ovary (CHO) cells purposely grown under conditions that enhanced the content of HM-glycans. The presence of an elevated level of mannose in the culture of the producer cells as well as supplementation with a specific mannosidase inhibitor resulted in purified mAbs containing significantly higher levels of HM-glycans than would be expected normally under standard conditions. We showed that HM-glycans in the purified mAbs were reduced significantly by the application of lectin chromatography. Analysis by both UPLC using fluorescence detection as well as by mass spectrometry showed that the removal of the HM-glycoforms was selective and did not affect the profile of the remaining glycoforms. This method has potential for use in large-scale biomanufacturing for the reduction or elimination of HM-glycoforms of mAbs.
{"title":"Reduction of high mannose glycoforms from monoclonal antibodies by affinity chromatography using a recombinant prokaryotic lectin","authors":"Elizabeth Matthews , Neha Tushar Dalvi , Michael Butler","doi":"10.1016/j.jbiotec.2025.09.008","DOIUrl":"10.1016/j.jbiotec.2025.09.008","url":null,"abstract":"<div><div>Glycan profiles of monoclonal antibodies are critical quality attributes for their use as therapeutic agents. However, the control of glycosylation can prove difficult during large-scale manufacture. An on-going problem is the increase in high-mannose glycans (HM) that can occur unexpectedly under cell culture conditions that disrupt the glycosylation pathway. We have developed a method for removal of high-mannose glycans from a heterogeneous mixture of mAb glycoforms purified from a bioprocess. This involves the use of a prokaryotic lectin (RPL-Man2) bound to agarose beads to selectively bind reversibly to mAbs carrying HM-glycans. The unbound flow-through from this step contains the remainder of the mAb preparation with a reduced HM-glycan content. In this paper we demonstrate the application of this method using two distinct antibodies derived from Chinese hamster ovary (CHO) cells purposely grown under conditions that enhanced the content of HM-glycans. The presence of an elevated level of mannose in the culture of the producer cells as well as supplementation with a specific mannosidase inhibitor resulted in purified mAbs containing significantly higher levels of HM-glycans than would be expected normally under standard conditions. We showed that HM-glycans in the purified mAbs were reduced significantly by the application of lectin chromatography. Analysis by both UPLC using fluorescence detection as well as by mass spectrometry showed that the removal of the HM-glycoforms was selective and did not affect the profile of the remaining glycoforms. This method has potential for use in large-scale biomanufacturing for the reduction or elimination of HM-glycoforms of mAbs.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 158-167"},"PeriodicalIF":3.9,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102709","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}
Pub Date : 2025-09-17DOI: 10.1016/j.jbiotec.2025.09.007
Siqi Ma, Chang Liu, Chenfeng Ma, Shuguang Wang
Urine recovery has been achieved through physical-chemical technologies on space station presently, but it requires harsh operation conditions and careful equipment maintenance. Here, a novel microreactor that combined microchannel and urease-crosslinked enzyme aggregates (urease-CLEAs) was prepared to mildly reclaim water and nitrogen nutrient from urine. First, the material that was suitable to make microchannel reactor and immobilize urease-CLEAs was selected from five organic materials, and poly(methyl methacrylate) was the best one. Second, the immobilization conditions for urease-CLEAs were optimized. Microchannel reactor with immobilized urease-CLEAs (CLEAs-microreactor) showed the highest activity of 0.54 U/cm2 under the optimal conditions of precipitant 20 % acetone, crosslinker 2.0 % glutaraldehyde, and 2.0-hour precipitation/crosslinking. CLEAs-microreactor showed good stability of pH, thermal, and storage compared with the microchannel reactor with immobilized free-urease (Free-microreactor). The efficiency of CLEAs-microreactor reached the highest of 101.8 μmol/h, which was 2.24-fold of Free-microreactor. This study provides an alternative technology for the recovery of water and nitrogen nutrient from urine on space station.
{"title":"Preparation of enzymatic microchannel reactor with immobilized urease-crosslinked enzyme aggregates and its performance","authors":"Siqi Ma, Chang Liu, Chenfeng Ma, Shuguang Wang","doi":"10.1016/j.jbiotec.2025.09.007","DOIUrl":"10.1016/j.jbiotec.2025.09.007","url":null,"abstract":"<div><div>Urine recovery has been achieved through physical-chemical technologies on space station presently, but it requires harsh operation conditions and careful equipment maintenance. Here, a novel microreactor that combined microchannel and urease-crosslinked enzyme aggregates (urease-CLEAs) was prepared to mildly reclaim water and nitrogen nutrient from urine. First, the material that was suitable to make microchannel reactor and immobilize urease-CLEAs was selected from five organic materials, and poly(methyl methacrylate) was the best one. Second, the immobilization conditions for urease-CLEAs were optimized. Microchannel reactor with immobilized urease-CLEAs (CLEAs-microreactor) showed the highest activity of 0.54 U/cm<sup>2</sup> under the optimal conditions of precipitant 20 % acetone, crosslinker 2.0 % glutaraldehyde, and 2.0-hour precipitation/crosslinking. CLEAs-microreactor showed good stability of pH, thermal, and storage compared with the microchannel reactor with immobilized free-urease (Free-microreactor). The efficiency of CLEAs-microreactor reached the highest of 101.8 μmol/h, which was 2.24-fold of Free-microreactor. This study provides an alternative technology for the recovery of water and nitrogen nutrient from urine on space station.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 142-150"},"PeriodicalIF":3.9,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145091458","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}
Pub Date : 2025-09-16DOI: 10.1016/j.jbiotec.2025.09.006
Yinhao Gao , Yazi Zhou , Liqian Wang , Na Zhang , Weishuai Qin , Wu Meng , Cuixia Zhou
Bacillus licheniformis is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the B. licheniformis strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in acoR and budC, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of acoR and budC increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of budC. Heterologous expression of acoR from B. licheniformis 2709 in both the wild type and acoR knockout mutant strongly inhibited acetoin accumulation compared to native acoR, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of budC restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of alsD in the acoR mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of bdhA and gldA increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of acoR and budC in acetoin and 2,3-BD metabolism, establishing a "knockout-overexpression" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.
{"title":"Synergistic strategy for high-yield 2,3-butanediol and acetoin production in Bacillus licheniformis MW03 based on metabolic engineering","authors":"Yinhao Gao , Yazi Zhou , Liqian Wang , Na Zhang , Weishuai Qin , Wu Meng , Cuixia Zhou","doi":"10.1016/j.jbiotec.2025.09.006","DOIUrl":"10.1016/j.jbiotec.2025.09.006","url":null,"abstract":"<div><div><em>Bacillus licheniformis</em> is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the <em>B. licheniformis</em> strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in <em>acoR</em> and <em>budC</em>, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of <em>acoR</em> and <em>budC</em> increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of <em>budC</em>. Heterologous expression of <em>acoR</em> from <em>B. licheniformis</em> 2709 in both the wild type and <em>acoR</em> knockout mutant strongly inhibited acetoin accumulation compared to native <em>acoR</em>, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of <em>budC</em> restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of <em>alsD</em> in the <em>acoR</em> mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of <em>bdhA</em> and <em>gldA</em> increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of <em>acoR</em> and <em>budC</em> in acetoin and 2,3-BD metabolism, establishing a \"knockout-overexpression\" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 232-243"},"PeriodicalIF":3.9,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145080838","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}
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