Pub Date : 2025-11-12DOI: 10.1016/j.jbiotec.2025.11.011
Mercedes Peña , Patricia Gómez-Villegas , Jose Prados , Consolación Melguizo , Carla C.C.R. de Carvalho
Archaea and bacteria are the most studied extremophiles, but fungi also demonstrate remarkable tolerance, particularly in hypersaline environments such as solar salterns. Among salt-adapted fungi, black yeasts have been shown to be adapted to such environments, having developed defense mechanisms such as the production of melanin, a pigment that plays a crucial role in environmental stress protection. Melanin is a complex, high-molecular-weight polymer widely found across biological kingdoms. During the isolation of microorganisms from samples collected in crystallization ponds in a saltern, a black yeast was found able to produce significant amounts of melanin. The yeast was identified as belonging to the species Neophaeotheca triangularis. The aim of this study was to optimize the cultivation conditions of the strain, to characterize the produced melanin, and to assess its biological activities, including its antitumor and antioxidant properties.
{"title":"Halotolerant black yeast Neophaeotheca triangularis as a source of melanin","authors":"Mercedes Peña , Patricia Gómez-Villegas , Jose Prados , Consolación Melguizo , Carla C.C.R. de Carvalho","doi":"10.1016/j.jbiotec.2025.11.011","DOIUrl":"10.1016/j.jbiotec.2025.11.011","url":null,"abstract":"<div><div>Archaea and bacteria are the most studied extremophiles, but fungi also demonstrate remarkable tolerance, particularly in hypersaline environments such as solar salterns. Among salt-adapted fungi, black yeasts have been shown to be adapted to such environments, having developed defense mechanisms such as the production of melanin, a pigment that plays a crucial role in environmental stress protection. Melanin is a complex, high-molecular-weight polymer widely found across biological kingdoms. During the isolation of microorganisms from samples collected in crystallization ponds in a saltern, a black yeast was found able to produce significant amounts of melanin. The yeast was identified as belonging to the species <em>Neophaeotheca triangularis</em>. The aim of this study was to optimize the cultivation conditions of the strain, to characterize the produced melanin, and to assess its biological activities, including its antitumor and antioxidant properties.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 10-22"},"PeriodicalIF":3.9,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145523618","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 : 2025-11-12DOI: 10.1016/j.jbiotec.2025.11.010
Beini Sun , Gengqiang Cao , Tian Gan , Yue Wang , Rumeng Qu , Lin Hu , Yuan Chang , Min Hu , Xiaoping Wang , Tongsheng Chen
Precise targeted drug screening is the key to improve the efficiency of tumour targeted therapy. This report presents a live cell FRET two-hybrid assay-based targeted drug screening method (FRET-HBTDS). In FRET-HBTDS, the cells co-expressing donor- and acceptor-labelled targets were cultured in 96-well plates, quantitative FRET imaging was then performed on a self-built automated FRET microscope (FRETscope) well-by-well, and FRET two-hybrid assay was used to obtain the maximum donor-centre FRET efficiency (EDmax), maximum acceptor-centre FRET efficiency (EAmax) and stoichiometric ratios (NA/ND). The FRET-HBTDS method was performed on the FRETscope with a 20 × objective for the cells co-expressing CFP-Bcl-xL and YFP-Bak to assess the action of eight compounds (A1331852, S63845, AC, DSF/Cu, Met, REGO, SOFA, ABT199) on the interaction between Bcl-xL and Bak. After 7 h of treatment with these compounds respectively, only the A1331852 group showed significantly lower EDmax and EAmax compared to the control group, and a significant increase in NA/ND, suggesting that A1331852 unlocks the direct interaction between Bcl-xL and Bak, thus releasing Bak to induce cell death. In addition, the NA/ND of the DSF/Cu group was significantly higher than of the control group, suggesting that DSF/Cu altered the stoichiometry of the Bcl-xL-Bak complex. Our data firmly demonstrate that A1331852 unlocks the binding state of Bcl-xL and Bak, while DSF/Cu modifies the structure of the Bcl-xL-Bak complex. These findings demonstrate that FRET-HBTDS can be used to assess the efficacy of a drug by revealing the binding state and complex molecular structure of the target proteins using FRET technology in living cells, which may be a potential targeted drug screening method.
{"title":"FRET two-hybrid assay-based target drug screening in living cells","authors":"Beini Sun , Gengqiang Cao , Tian Gan , Yue Wang , Rumeng Qu , Lin Hu , Yuan Chang , Min Hu , Xiaoping Wang , Tongsheng Chen","doi":"10.1016/j.jbiotec.2025.11.010","DOIUrl":"10.1016/j.jbiotec.2025.11.010","url":null,"abstract":"<div><div>Precise targeted drug screening is the key to improve the efficiency of tumour targeted therapy. This report presents a live cell FRET two-hybrid assay-based targeted drug screening method (FRET-HBTDS). In FRET-HBTDS, the cells co-expressing donor- and acceptor-labelled targets were cultured in 96-well plates, quantitative FRET imaging was then performed on a self-built automated FRET microscope (FRETscope) well-by-well, and FRET two-hybrid assay was used to obtain the maximum donor-centre FRET efficiency (<em>E</em><sub><em>Dmax</em></sub>), maximum acceptor-centre FRET efficiency (<em>E</em><sub><em>Amax</em></sub>) and stoichiometric ratios (<em>N</em><sub><em>A</em></sub><em>/N</em><sub><em>D</em></sub>). The FRET-HBTDS method was performed on the FRETscope with a 20 × objective for the cells co-expressing CFP-Bcl-xL and YFP-Bak to assess the action of eight compounds (A1331852, S63845, AC, DSF/Cu, Met, REGO, SOFA, ABT199) on the interaction between Bcl-xL and Bak. After 7 h of treatment with these compounds respectively, only the A1331852 group showed significantly lower <em>E</em><sub><em>Dmax</em></sub> and <em>E</em><sub><em>Amax</em></sub> compared to the control group, and a significant increase in <em>N</em><sub><em>A</em></sub><em>/N</em><sub><em>D</em></sub>, suggesting that A1331852 unlocks the direct interaction between Bcl-xL and Bak, thus releasing Bak to induce cell death. In addition, the <em>N</em><sub><em>A</em></sub><em>/N</em><sub><em>D</em></sub> of the DSF/Cu group was significantly higher than of the control group, suggesting that DSF/Cu altered the stoichiometry of the Bcl-xL-Bak complex. Our data firmly demonstrate that A1331852 unlocks the binding state of Bcl-xL and Bak, while DSF/Cu modifies the structure of the Bcl-xL-Bak complex. These findings demonstrate that FRET-HBTDS can be used to assess the efficacy of a drug by revealing the binding state and complex molecular structure of the target proteins using FRET technology in living cells, which may be a potential targeted drug screening method.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 1-9"},"PeriodicalIF":3.9,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145523543","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 : 2025-11-08DOI: 10.1016/j.jbiotec.2025.11.003
Sangavi Sivananthan , S. Ameer Ahmed , Ammaar M. Baig , Varsha Bhakta , William P. Sheffield
{"title":"Corrigendum to “Substitution of reactive centre loop residues from C1 esterase inhibitor increases the inhibitory specificity of alpha-1 antitrypsin for plasma kallikrein” [J. Biotechnol. 405 (2025) 205–214]","authors":"Sangavi Sivananthan , S. Ameer Ahmed , Ammaar M. Baig , Varsha Bhakta , William P. Sheffield","doi":"10.1016/j.jbiotec.2025.11.003","DOIUrl":"10.1016/j.jbiotec.2025.11.003","url":null,"abstract":"","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Page 182"},"PeriodicalIF":3.9,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145481187","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 : 2025-11-07DOI: 10.1016/j.jbiotec.2025.11.009
Kang Zhao , Sha-Sha Zhao , Quan-Wei Liu , Zhen Liu
To address the challenge of lignin removal while preserving cellulose in lignocellulosic biomass valorization, this study developed a novel ternary deep eutectic solvent (DES) system composed of triethylbenzyl ammonium chloride (T, as hydrogen bond acceptor), copper chloride (Lewis acid catalyst), and ethylene glycol (EG, hydrogen bond donor) for microwave-assisted pretreatment of corn straw. The DES was designed to leverage synergistic effects between Lewis acid catalysis and hydrogen bond disruption for selective lignin extraction. Optimization via Box-Behnken experimental design revealed that optimal delignification (96.52 %) and hemicellulose removal (94.60 %) with 94.79 % cellulose retention were achieved under microwave conditions of 130°C, 40 min, liquid-solid ratio 13.1, and CuCl₂/T molar ratio 0.198. Comparative characterization of raw and pretreated biomass using SEM, FT-IR, and XRD demonstrated that T-CuCl₂-EG disrupted the lignocellulose matrix by breaking ether/ester bonds in lignin-carbohydrate complexes, selectively solubilizing lignin/hemicellulose while preserving cellulose crystallinity. This work provides a sustainable and efficient strategy for biomass fractionation, with potential applications in biorefineries.
{"title":"Ternary deep eutectic solvent (T-CuCl2-EG) for lignin removal from corn straw","authors":"Kang Zhao , Sha-Sha Zhao , Quan-Wei Liu , Zhen Liu","doi":"10.1016/j.jbiotec.2025.11.009","DOIUrl":"10.1016/j.jbiotec.2025.11.009","url":null,"abstract":"<div><div>To address the challenge of lignin removal while preserving cellulose in lignocellulosic biomass valorization, this study developed a novel ternary deep eutectic solvent (DES) system composed of triethylbenzyl ammonium chloride (T, as hydrogen bond acceptor), copper chloride (Lewis acid catalyst), and ethylene glycol (EG, hydrogen bond donor) for microwave-assisted pretreatment of corn straw. The DES was designed to leverage synergistic effects between Lewis acid catalysis and hydrogen bond disruption for selective lignin extraction. Optimization via Box-Behnken experimental design revealed that optimal delignification (96.52 %) and hemicellulose removal (94.60 %) with 94.79 % cellulose retention were achieved under microwave conditions of 130°C, 40 min, liquid-solid ratio 13.1, and CuCl₂/T molar ratio 0.198. Comparative characterization of raw and pretreated biomass using SEM, FT-IR, and XRD demonstrated that T-CuCl₂-EG disrupted the lignocellulose matrix by breaking ether/ester bonds in lignin-carbohydrate complexes, selectively solubilizing lignin/hemicellulose while preserving cellulose crystallinity. This work provides a sustainable and efficient strategy for biomass fractionation, with potential applications in biorefineries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 195-201"},"PeriodicalIF":3.9,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145481162","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}
Microalgal stress-related responses to nitrogen (N) deprivation are determined by genetically defined metabolic changes, which are further influenced by cultivation and nutritional conditions. In this study, we evaluated the metabolic responses of five chlorophyte strains to varying N concentrations. The strains included Chlamydomonas reinhardtii and Scenedesmus obliquus, which accumulated higher levels of starch, and Monoraphidium sp., Chlamydomonas sp., and Chlorella vulgaris, characterized by differences in both the levels and compositions of accumulated lipids. Under N-starved condition, all strains exhibited increased starch and neutral lipid content, with strain-specific variations. In this treatment, the content of branched-chain amino acids decreased in Monoraphidium sp., likely due to amino acid catabolism associated with the autophagy and triacylglycerol (TAG) accumulation. Furthermore, we observed a remodeling of lipid classes in S. obliquus, C. vulgaris, Chlamydomonas sp., and Monoraphidium sp., including reductions in digalactosyl diacylglycerol, monogalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol, accompanied by an increase in TAGs. Under N-starvation several fatty acids of C. vulgaris, Chlamydomonas sp., and C reinhardtii, including α-linolenic acid (C18:3), oleic acid (C18:1), and linoleic acid (C18:2) increase. In contrast, Monoraphidium sp. accumulated long-chain fatty acids such as lumequic acid (C30:1), nervonic acid (C24:1), docosanoic acid (C22:0), and erucic acid (C22:1). Collectively, these results indicate that chlorphyte strains exhibit distinct metabolic responses to N depletion in the medium. These findings highlight the importance of strain-specific assessments when developing the N management strategies for microalgal cultivation and biotechnological applications.
{"title":"Differential metabolic reprogramming due to nitrogen starvation leads to remobilization of storage metabolites in microalgal strains","authors":"Lidiane Covell , Mariana Machado , Marcelo Gomes Marçal Vieira Vaz , Auxiliadora Oliveira Martins , Carolina Cassano Monte Bello , Umarah Mubeen , Dilson Novais Rocha , Marcio Arêdes Martins , Camila Caldana , Wagner L. Araújo , Adriano Nunes-Nesi","doi":"10.1016/j.jbiotec.2025.11.008","DOIUrl":"10.1016/j.jbiotec.2025.11.008","url":null,"abstract":"<div><div>Microalgal stress-related responses to nitrogen (N) deprivation are determined by genetically defined metabolic changes, which are further influenced by cultivation and nutritional conditions. In this study, we evaluated the metabolic responses of five chlorophyte strains to varying N concentrations. The strains included <em>Chlamydomonas reinhardtii</em> and <em>Scenedesmus obliquus</em>, which accumulated higher levels of starch, and <em>Monoraphidium</em> sp., <em>Chlamydomonas</em> sp., and <em>Chlorella vulgaris</em>, characterized by differences in both the levels and compositions of accumulated lipids. Under N-starved condition, all strains exhibited increased starch and neutral lipid content, with strain-specific variations. In this treatment, the content of branched-chain amino acids decreased in <em>Monoraphidium</em> sp., likely due to amino acid catabolism associated with the autophagy and triacylglycerol (TAG) accumulation. Furthermore, we observed a remodeling of lipid classes in <em>S. obliquus</em>, <em>C. vulgaris</em>, <em>Chlamydomonas</em> sp., and <em>Monoraphidium</em> sp., including reductions in digalactosyl diacylglycerol, monogalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol, accompanied by an increase in TAGs. Under N-starvation several fatty acids of <em>C. vulgaris</em>, <em>Chlamydomonas</em> sp., and <em>C reinhardtii</em>, including α-linolenic acid (C18:3), oleic acid (C18:1), and linoleic acid (C18:2) increase. In contrast, <em>Monoraphidium</em> sp. accumulated long-chain fatty acids such as lumequic acid (C30:1), nervonic acid (C24:1), docosanoic acid (C22:0), and erucic acid (C22:1). Collectively, these results indicate that chlorphyte strains exhibit distinct metabolic responses to N depletion in the medium. These findings highlight the importance of strain-specific assessments when developing the N management strategies for microalgal cultivation and biotechnological applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 231-246"},"PeriodicalIF":3.9,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482251","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 : 2025-11-06DOI: 10.1016/j.jbiotec.2025.11.001
Die Zhao , Chunji Li , Nan Zeng , Dandan Wang , Anqi Zeng , Guohui Yu , Ning Zhang , Bingxue Li
Carotenoids are bioactive pigments widely used in food, pharmaceutical, and cosmetic industries. Rhodosporidiobolus odoratus co-produces commercially significant carotenoids, including β-carotene, torulene, and torularhodin. This study aimed to reveal how carotenoid biosynthesis in R. odoratus XQR, optimally grown at 20 °C, responds to low (10 °C) and high (30 °C) temperatures. After five days, total carotenoids reached 118.55 μg/g DCW and 0.40 μg/mL at 30 °C, ∼3-fold higher than the 20 °C control (41.25 μg/g DCW and 0.14 μg/mL). Torularhodin showed a pronounced increase at 30 °C (37.26 μg/g DCW and 0.12 μg/mL), ∼9-fold above the control (4.06 μg/g DCW and 0.014 μg/mL). At 10 °C, total carotenoids declined to 26.09 μg/g DCW, with a slight, non-significant rise in volumetric titer to 0.17 μg/mL. High temperature elevated reactive oxygen species (ROS) and superoxide dismutase (SOD) activity while reducing total protein, whereas low temperature maintained stable ROS, induced moderate SOD, and caused protein decline; catalase (CAT) activity changed minimally under both conditions. Our integrated data suggest that high temperature promotes carotenoid overaccumulation through upregulation of key terpenoid backbone biosynthetic genes (HMGCS, hmgA, and mvaD) and carotenogenic genes (crtYB, crtI, crtZ, and crtA), coupled with a metabolic shift that enhanced precursor supply. These coordinated responses explain torularhodin-dominant accumulation and differ from related yeasts, suggesting species-specific regulation. This work provides new mechanistic insights into temperature-driven carotenogenesis in R. odoratus XQR and highlights targets for metabolic engineering.
{"title":"High temperature drives torularhodin-dominant carotenoid overaccumulation in Rhodosporidiobolus odoratus XQR via coordinated transcriptomic–metabolomic reprogramming","authors":"Die Zhao , Chunji Li , Nan Zeng , Dandan Wang , Anqi Zeng , Guohui Yu , Ning Zhang , Bingxue Li","doi":"10.1016/j.jbiotec.2025.11.001","DOIUrl":"10.1016/j.jbiotec.2025.11.001","url":null,"abstract":"<div><div>Carotenoids are bioactive pigments widely used in food, pharmaceutical, and cosmetic industries. <em>Rhodosporidiobolus odoratus</em> co-produces commercially significant carotenoids, including β-carotene, torulene, and torularhodin. This study aimed to reveal how carotenoid biosynthesis in <em>R. odoratus</em> XQR, optimally grown at 20 °C, responds to low (10 °C) and high (30 °C) temperatures. After five days, total carotenoids reached 118.55 μg/g DCW and 0.40 μg/mL at 30 °C, ∼3-fold higher than the 20 °C control (41.25 μg/g DCW and 0.14 μg/mL). Torularhodin showed a pronounced increase at 30 °C (37.26 μg/g DCW and 0.12 μg/mL), ∼9-fold above the control (4.06 μg/g DCW and 0.014 μg/mL). At 10 °C, total carotenoids declined to 26.09 μg/g DCW, with a slight, non-significant rise in volumetric titer to 0.17 μg/mL. High temperature elevated reactive oxygen species (ROS) and superoxide dismutase (SOD) activity while reducing total protein, whereas low temperature maintained stable ROS, induced moderate SOD, and caused protein decline; catalase (CAT) activity changed minimally under both conditions. Our integrated data suggest that high temperature promotes carotenoid overaccumulation through upregulation of key terpenoid backbone biosynthetic genes (<em>HMGCS</em>, <em>hmgA</em>, and <em>mvaD</em>) and carotenogenic genes (<em>crtYB</em>, <em>crtI</em>, <em>crtZ</em>, and <em>crtA</em>), coupled with a metabolic shift that enhanced precursor supply. These coordinated responses explain torularhodin-dominant accumulation and differ from related yeasts, suggesting species-specific regulation. This work provides new mechanistic insights into temperature-driven carotenogenesis in <em>R. odoratus</em> XQR and highlights targets for metabolic engineering.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 215-230"},"PeriodicalIF":3.9,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145476626","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 : 2025-11-05DOI: 10.1016/j.jbiotec.2025.11.005
Shuyao Ma , Yufan Jing , Fangyuan Liu , Xiaolong Zheng , Xing Zhang , Meng Qiao
As a non-conventional oleaginous yeast, Yarrowia lipolytica (Y. lipolytica) has emerged as a promising host for carotenoid production due to its robust metabolism capabilities and lipids accumulate ability. The metabolic engineering of Y. lipolytica necessitates genetic expression tools, genome editing technologies, and systematic strategies for optimizing the global biosynthesis pathway. In this review, we summarize key genetic regulatory elements and gene editing tools that have been employed to enhance carotenoid biosynthesis in Y. lipolytica. We also examine representative metabolic engineering cases involving the production of carotenoids, such as lycopene, β-carotene, astaxanthin, and lutein, by Y. lipolytica with a focus on precursor pathway enhancement, subcellular compartmentalization, as well as enzyme modification and expression tuning. Furthermore, perspectives are provided on future strategies to engineer high-performance Y. lipolytica strains for industrial carotenoid production.
{"title":"Metabolic engineering of carotenoid biosynthesis in Yarrowia lipolytica","authors":"Shuyao Ma , Yufan Jing , Fangyuan Liu , Xiaolong Zheng , Xing Zhang , Meng Qiao","doi":"10.1016/j.jbiotec.2025.11.005","DOIUrl":"10.1016/j.jbiotec.2025.11.005","url":null,"abstract":"<div><div>As a non-conventional oleaginous yeast, <em>Yarrowia lipolytica</em> (<em>Y. lipolytica</em>) has emerged as a promising host for carotenoid production due to its robust metabolism capabilities and lipids accumulate ability. The metabolic engineering of <em>Y. lipolytica</em> necessitates genetic expression tools, genome editing technologies, and systematic strategies for optimizing the global biosynthesis pathway. In this review, we summarize key genetic regulatory elements and gene editing tools that have been employed to enhance carotenoid biosynthesis in <em>Y. lipolytica</em>. We also examine representative metabolic engineering cases involving the production of carotenoids, such as lycopene, β-carotene, astaxanthin, and lutein, by <em>Y. lipolytica</em> with a focus on precursor pathway enhancement, subcellular compartmentalization, as well as enzyme modification and expression tuning. Furthermore, perspectives are provided on future strategies to engineer high-performance <em>Y. lipolytica</em> strains for industrial carotenoid production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 202-214"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471038","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 : 2025-11-05DOI: 10.1016/j.jbiotec.2025.11.004
Robert Klausser , Lukas Veiter , Julian Kopp , Nikolaus Hammerschmidt , Tiffany Frierss , Florian Gisperg , Mohamed Elshazly , Eva Prada Brichtova , Michael Martinetz , Martin Voigtmann , Oliver Spadiut
Therapeutic proteins are often produced intracellularly in the bacterium Escherichia coli in aggregated form, known as inclusion bodies, which must be solubilized and refolded to recover the biologically active product. However, the industrially established refolding method of batch dilution is still carried out at low protein concentrations, leading to high water consumption, large equipment footprints, and poor sustainability. An underexplored way to increase refolding yields and thereby enable higher product concentrations after refolding is the optimization of cell lysis and inclusion body washing. In this study, we developed and evaluated a “recursive high pressure homogenization” approach, wherein wash steps and additional homogenization cycles were combined to enhance the final purity of inclusion bodies before solubilization. The degree of cell lysis, nucleic acid release and final inclusion body purity were compared to conventional “linear washing”, where all homogenization cycles are completed prior to the washing of inclusion bodies. In total, seven process variations were compared and the resulting batches of inclusion bodies were solubilized and refolded to investigate the effect of early downstream processing on the product concentration in the final refolds. For the most efficient process variant, the recursive high pressure homogenization protocol led to the highest measured product concentration of 855 mg/L after refolding. Thus, the proposed recursive high pressure homogenization approach led to an estimated 18 % reduction of CO2 footprint caused by urea, and increased the product yield per biomass from 5.17 g/kg to 7.84 g/kg compared to its linear wash counterpart — without introducing non-standard equipment or chemicals.
{"title":"Increased purity and refolding yield of bacterial inclusion bodies by recursive high pressure homogenization","authors":"Robert Klausser , Lukas Veiter , Julian Kopp , Nikolaus Hammerschmidt , Tiffany Frierss , Florian Gisperg , Mohamed Elshazly , Eva Prada Brichtova , Michael Martinetz , Martin Voigtmann , Oliver Spadiut","doi":"10.1016/j.jbiotec.2025.11.004","DOIUrl":"10.1016/j.jbiotec.2025.11.004","url":null,"abstract":"<div><div>Therapeutic proteins are often produced intracellularly in the bacterium <em>Escherichia coli</em> in aggregated form, known as inclusion bodies, which must be solubilized and refolded to recover the biologically active product. However, the industrially established refolding method of batch dilution is still carried out at low protein concentrations, leading to high water consumption, large equipment footprints, and poor sustainability. An underexplored way to increase refolding yields and thereby enable higher product concentrations after refolding is the optimization of cell lysis and inclusion body washing. In this study, we developed and evaluated a “recursive high pressure homogenization” approach, wherein wash steps and additional homogenization cycles were combined to enhance the final purity of inclusion bodies before solubilization. The degree of cell lysis, nucleic acid release and final inclusion body purity were compared to conventional “linear washing”, where all homogenization cycles are completed prior to the washing of inclusion bodies. In total, seven process variations were compared and the resulting batches of inclusion bodies were solubilized and refolded to investigate the effect of early downstream processing on the product concentration in the final refolds. For the most efficient process variant, the recursive high pressure homogenization protocol led to the highest measured product concentration of 855 mg/L after refolding. Thus, the proposed recursive high pressure homogenization approach led to an estimated 18 % reduction of CO<sub>2</sub> footprint caused by urea, and increased the product yield per biomass from 5.17 g/kg to 7.84 g/kg compared to its linear wash counterpart — without introducing non-standard equipment or chemicals.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 183-194"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471041","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 : 2025-11-05DOI: 10.1016/j.jbiotec.2025.11.002
Rémi Hocq , Gwladys Chartier, Nicolas Lopes Ferreira, François Wasels
The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.
{"title":"CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii","authors":"Rémi Hocq , Gwladys Chartier, Nicolas Lopes Ferreira, François Wasels","doi":"10.1016/j.jbiotec.2025.11.002","DOIUrl":"10.1016/j.jbiotec.2025.11.002","url":null,"abstract":"<div><div>The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from <em>Listeria monocytogenes</em> to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant <em>Clostridium beijerinckii</em> DSM 6423. While deletion of <em>upp</em> alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 165-169"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471016","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 : 2025-11-05DOI: 10.1016/j.jbiotec.2025.11.006
Dong Yu , Lin Zhu , Ya-Xue Ding , Li-Jing Mao , Min Xiong , Xiao-Xuan Jin , Yujie Ma , Feng-Qing Wang , Liang-Bin Xiong
5-aminolevulinic acid (ALA), a non‑proteinogenic δ‑amino acid, is a versatile compound with applications as a tumor-sensitizing agent in photodynamic therapy and a plant biostimulant that enhances stress tolerance and photosynthetic efficiency. This study aimed to leverage the endogenous cryptic plasmids pMUT1 and pMUT2 of the probiotic Escherichia coli Nissle 1917 (EcN) to construct a self-sufficient strain with autonomous ALA biosynthesis capability, eliminating dependencies on exogenous plasmids or chemical inducers. A synthetic operon was designed on a re-engineered cryptic plasmid to express core genes of the C5 pathway (gltX, hemA, and hemL). Systematic evaluation demonstrated that overexpression of gltX alone resulted in minimal ALA accumulation (6.8 mg/L). In contrast, coordinated co-expression of hemA and hemL significantly increased ALA titers to 854.5 mg/L, highlighting their synergistic role in channeling carbon flux through the C5 pathway in EcN. Subsequent optimization of the hemA‑hemL cassette further elevated production to 925.1 mg/L. Notably, this enhanced ALA synthesis perturbed the cellular NADP+ /NADPH balance. To address this, we integrated pos5, encoding a NADP⁺‑dependent transhydrogenase, into the endogenous plasmid, enabling in situ NADPH regeneration and boosting ALA titers to 1306.8 mg/L. Scale up to a 5-L bioreactor with fed-batch cultivation and controlled glycerol feeding achieved an ALA titer of 3372.5 ± 162.7 mg/L at 108 h. This endogenous plasmid-centric approach establishes an inducer-free, antibiotic-independent microbial cell factory, positioning EcN as a universal platform for ALA production with potential in biomedical and agricultural applications.
{"title":"Engineering of endogenous plasmids in probiotic Escherichia coli Nissle 1917 for autonomous accumulation of 5‑aminolevulinic acid","authors":"Dong Yu , Lin Zhu , Ya-Xue Ding , Li-Jing Mao , Min Xiong , Xiao-Xuan Jin , Yujie Ma , Feng-Qing Wang , Liang-Bin Xiong","doi":"10.1016/j.jbiotec.2025.11.006","DOIUrl":"10.1016/j.jbiotec.2025.11.006","url":null,"abstract":"<div><div>5-aminolevulinic acid (ALA), a non‑proteinogenic δ‑amino acid, is a versatile compound with applications as a tumor-sensitizing agent in photodynamic therapy and a plant biostimulant that enhances stress tolerance and photosynthetic efficiency. This study aimed to leverage the endogenous cryptic plasmids pMUT1 and pMUT2 of the probiotic <em>Escherichia coli</em> Nissle 1917 (EcN) to construct a self-sufficient strain with autonomous ALA biosynthesis capability, eliminating dependencies on exogenous plasmids or chemical inducers. A synthetic operon was designed on a re-engineered cryptic plasmid to express core genes of the C5 pathway (<em>gltX</em>, <em>hemA</em>, and <em>hemL</em>). Systematic evaluation demonstrated that overexpression of <em>gltX</em> alone resulted in minimal ALA accumulation (6.8 mg/L). In contrast, coordinated co-expression of <em>hemA</em> and <em>hemL</em> significantly increased ALA titers to 854.5 mg/L, highlighting their synergistic role in channeling carbon flux through the C5 pathway in EcN. Subsequent optimization of the <em>hemA‑hemL</em> cassette further elevated production to 925.1 mg/L. Notably, this enhanced ALA synthesis perturbed the cellular NADP<sup>+</sup> /NADPH balance. To address this, we integrated <em>pos5</em>, encoding a NADP⁺‑dependent transhydrogenase, into the endogenous plasmid, enabling <em>in situ</em> NADPH regeneration and boosting ALA titers to 1306.8 mg/L. Scale up to a 5-L bioreactor with fed-batch cultivation and controlled glycerol feeding achieved an ALA titer of 3372.5 ± 162.7 mg/L at 108 h. This endogenous plasmid-centric approach establishes an inducer-free, antibiotic-independent microbial cell factory, positioning EcN as a universal platform for ALA production with potential in biomedical and agricultural applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 157-164"},"PeriodicalIF":3.9,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471033","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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