Minicircle DNA vectors (MCs) are gene carriers with favorable characteristics for gene therapy and the production of recombinant cells using animal cells as hosts. Typically, MCs are prepared by inserting target gene fragments as an expression unit into a standard plasmid vector, followed by the removal of bacterial sequences. In this study, we report a method for MC preparation utilizing the Cre-loxP recombination system. By inserting the target gene fragments between two loxP sites introduced into the plasmid vector, the plasmid backbone can be removed through the action of Cre recombinase. We explored optimal reaction conditions for MC generation using Cre. Furthermore, site-specific knock-ins into the CHO cell genome were performed using the remaining loxP sequence in the generated MCs via the Cre-loxP reaction. When MCs were used as gene donors, significant improvements in gene integration efficiency and enhanced gene expression in CHO cells were observed compared with conventional plasmids. The MC preparation and gene knock-in techniques developed in this study are highly useful for CHO cell engineering.
{"title":"Preparation of minicircle DNA vectors for gene knock-in to mammalian cells","authors":"Yoshinori Kawabe, Makoto Hamaoka, Akio Kuno, Yusaku Yoshimura, Masamichi Kamihira","doi":"10.1016/j.jbiosc.2025.05.005","DOIUrl":"10.1016/j.jbiosc.2025.05.005","url":null,"abstract":"<div><div>Minicircle DNA vectors (MCs) are gene carriers with favorable characteristics for gene therapy and the production of recombinant cells using animal cells as hosts. Typically, MCs are prepared by inserting target gene fragments as an expression unit into a standard plasmid vector, followed by the removal of bacterial sequences. In this study, we report a method for MC preparation utilizing the Cre-<em>loxP</em> recombination system. By inserting the target gene fragments between two <em>loxP</em> sites introduced into the plasmid vector, the plasmid backbone can be removed through the action of Cre recombinase. We explored optimal reaction conditions for MC generation using Cre. Furthermore, site-specific knock-ins into the CHO cell genome were performed using the remaining <em>loxP</em> sequence in the generated MCs via the Cre-<em>loxP</em> reaction. When MCs were used as gene donors, significant improvements in gene integration efficiency and enhanced gene expression in CHO cells were observed compared with conventional plasmids. The MC preparation and gene knock-in techniques developed in this study are highly useful for CHO cell engineering.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 2","pages":"Pages 107-112"},"PeriodicalIF":2.3,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tissue-engineered three-dimensional (3D) skeletal muscles can be potentially used in contractile force-based phenotypic screening to elucidate the mechanisms of skeletal muscle dysfunction and develop preventive and therapeutic strategies. Human induced pluripotent stem cells (hiPSCs) expressing tetracycline-inducible myogenic differentiation 1 (MYOD1) are a promising cell source for construction of tissue-engineered skeletal muscles. Although we successfully constructed contractile tissues using these hiPSCs in a previous study, further improvements are required because of their weak contractile force and inefficient screening capabilities. In this study, we aimed to construct iPSC-derived muscle tissues with high contractile force using a 96-well scale microdevice that we had previously developed. To increase the contractile force, we optimized the time of supplementation of the transforming growth factor-β (TGF-β) inhibitor, SB431542 (SB), to identify culture conditions that enhance contractile force. The maximum contractile force with addition of SB was approximately five times greater than that without SB (58.45 ± 20.14 μN with SB compared to 11.64 ± 4.86 μN without SB). Various analyses, including immunostaining, transmission electron microscopy, gene expression analysis, and proteomics, revealed enhanced myotube differentiation and muscle tissue maturation in the presence of SB. Experiments using inhibitors indicated that TGFβ1, not myostatin, is partially involved in these effects. Furthermore, we confirmed that tissues constructed from iPSCs derived from patients with Duchenne muscular dystrophy also showed improved contractility following addition of SB. Therefore, iPSC-derived muscle tissues cultured with SB on the 96-well microdevice provide a promising platform for screening compounds that can ameliorate disease pathology.
{"title":"Construction of contractile human iPSC-derived skeletal muscle tissues on 96-well scale microdevices","authors":"Seitaro Nakamura , Yuhei Kamei , Ayumu Matsushima , Kazuki Yamamoto , Hirokazu Akiyama , Muhammad Irfanur Rashid , Yohei Okada , Tomoya Uchimura , Hidetoshi Sakurai , Hiroyuki Honda , Kazunori Shimizu","doi":"10.1016/j.jbiosc.2025.05.003","DOIUrl":"10.1016/j.jbiosc.2025.05.003","url":null,"abstract":"<div><div>Tissue-engineered three-dimensional (3D) skeletal muscles can be potentially used in contractile force-based phenotypic screening to elucidate the mechanisms of skeletal muscle dysfunction and develop preventive and therapeutic strategies. Human induced pluripotent stem cells (hiPSCs) expressing tetracycline-inducible myogenic differentiation 1 (MYOD1) are a promising cell source for construction of tissue-engineered skeletal muscles. Although we successfully constructed contractile tissues using these hiPSCs in a previous study, further improvements are required because of their weak contractile force and inefficient screening capabilities. In this study, we aimed to construct iPSC-derived muscle tissues with high contractile force using a 96-well scale microdevice that we had previously developed. To increase the contractile force, we optimized the time of supplementation of the transforming growth factor-β (TGF-β) inhibitor, SB431542 (SB), to identify culture conditions that enhance contractile force. The maximum contractile force with addition of SB was approximately five times greater than that without SB (58.45 ± 20.14 μN with SB compared to 11.64 ± 4.86 μN without SB). Various analyses, including immunostaining, transmission electron microscopy, gene expression analysis, and proteomics, revealed enhanced myotube differentiation and muscle tissue maturation in the presence of SB. Experiments using inhibitors indicated that TGFβ1, not myostatin, is partially involved in these effects. Furthermore, we confirmed that tissues constructed from iPSCs derived from patients with Duchenne muscular dystrophy also showed improved contractility following addition of SB. Therefore, iPSC-derived muscle tissues cultured with SB on the 96-well microdevice provide a promising platform for screening compounds that can ameliorate disease pathology.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 2","pages":"Pages 98-106"},"PeriodicalIF":2.3,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144181801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pseudozyma antarctica is a leaf-colonizing basidiomycetous yeast that produces a plastic-degrading enzyme (PaE) using xylose as an inducer and carbon source. Notably, glucose, a readily available carbon source, has not been utilized for PaE production. Herein, we identified two promoters of P. antarctica for stable induction of PaE gene transcription at high levels in commonly used yeast media with glucose, along with assessing the effect of copper supplementation. Through microarray analysis, the top five genes with high expression in glucose cultures were identified and their promoter activities were evaluated by reporter assay. The first and second genes were located 903 nucleotides apart on the P. antarctica chromosome and shared reciprocal inverted sequences as promoters, namely PaFRE1 and PaCTR1, respectively, owing to their weak similarity to FRE and CTR in Saccharomyces cerevisiae genome, which regulate the copper uptake. When P. antarctica was grown in three common media using glucose as a carbon source, both PaFRE1 and PaCTR1 induced high and stable expression and produced more heterologous artificial luciferase and endogenous PaE than that by the highly expressed actin promoter. Additionally, the activities of PaFRE1 and PaCTR1 promoters were suppressed by copper addition. Altogether, this study shows that these P. antarctica promoters can be used to produce various proteins such as PaE.
{"title":"Novel promoter of Pseudozyma antarctica exhibits stable glucose-mediated activation and copper-mediated suppression of flanking genes","authors":"Tohru Yarimizu , Atsuhiro Miura , Xiao-hong Cao , Takumi Tanaka , Tomotake Morita , Hitoshi Shimoi , Hirokazu Ueda , Hiroko Kitamoto","doi":"10.1016/j.jbiosc.2025.05.002","DOIUrl":"10.1016/j.jbiosc.2025.05.002","url":null,"abstract":"<div><div><em>Pseudozyma antarctica</em> is a leaf-colonizing basidiomycetous yeast that produces a plastic-degrading enzyme (PaE) using xylose as an inducer and carbon source. Notably, glucose, a readily available carbon source, has not been utilized for PaE production. Herein, we identified two promoters of <em>P. antarctica</em> for stable induction of PaE gene transcription at high levels in commonly used yeast media with glucose, along with assessing the effect of copper supplementation. Through microarray analysis, the top five genes with high expression in glucose cultures were identified and their promoter activities were evaluated by reporter assay. The first and second genes were located 903 nucleotides apart on the <em>P. antarctica</em> chromosome and shared reciprocal inverted sequences as promoters, namely Pa<em>FRE1</em> and Pa<em>CTR1</em>, respectively, owing to their weak similarity to <em>FRE</em> and <em>CTR</em> in <em>Saccharomyces cerevisiae</em> genome, which regulate the copper uptake. When <em>P. antarctica</em> was grown in three common media using glucose as a carbon source, both Pa<em>FRE1</em> and Pa<em>CTR1</em> induced high and stable expression and produced more heterologous artificial luciferase and endogenous PaE than that by the highly expressed actin promoter. Additionally, the activities of Pa<em>FRE1</em> and Pa<em>CTR1</em> promoters were suppressed by copper addition. Altogether, this study shows that these <em>P. antarctica</em> promoters can be used to produce various proteins such as PaE.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 2","pages":"Pages 66-72"},"PeriodicalIF":2.3,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144181644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-23DOI: 10.1016/j.jbiosc.2025.05.001
Rui Zhang, Keita Kanki
Decreased cell viability resulting from severe nutrient deprivation is a major obstacle in three-dimensional (3D) tissue construction. Therefore, technical improvements that prevent cell death in the core region of cell aggregates are desired for the development of large, thick tissues. We focused on the anti-glycolytic effects of resveratrol (RSV), a polyphenol known as a caloric restriction mimetic, and investigated its cytoprotective effects under glucose-deprived conditions in two-dimensional (2D) and 3D-cell culture systems using rat chondrocytes. In 2D culture, the low-glucose (LG, 0.5 mg/mL) condition caused time- and dose-dependent cell death in chondrocytes, whereas co-treatment with 50 μM RSV significantly restored cell viability under glucose deprivation. In RSV-treated cells, the expression levels of glycolytic genes (GLUT1, PKM, and LDHA) and glucose uptake were significantly downregulated, and phospho-AMPK levels were upregulated, indicating energy stress. RSV treatment restored the expression of extracellular matrix genes (COL1A1 and COL2A1), which were downregulated under the LG condition, and augmented the pro-chondrogenic effect of TGF-β1 and ascorbic acid. In a 3D-culture model, spheroids constructed with RSV-pretreated chondrocytes had a more viable core region than dimethyl-sulfoxide-treated control spheroids. TGF-β-induced cartilage maturation led these spheroids to develop larger and more viable tissues than control spheroids. These results suggested that glycolytic inhibition by RSV decreased chondrocyte glucose usage, thereby preventing cell death caused by glucose deprivation. Our findings provide useful information for improving cell viability under hyponutrition conditions and can be applied to 3D tissue construction.
{"title":"Glycolytic inhibition by resveratrol facilitates chondrocyte survival under glucose-deprived conditions and improves the viability of 3D-cultured cartilage tissue","authors":"Rui Zhang, Keita Kanki","doi":"10.1016/j.jbiosc.2025.05.001","DOIUrl":"10.1016/j.jbiosc.2025.05.001","url":null,"abstract":"<div><div>Decreased cell viability resulting from severe nutrient deprivation is a major obstacle in three-dimensional (3D) tissue construction. Therefore, technical improvements that prevent cell death in the core region of cell aggregates are desired for the development of large, thick tissues. We focused on the anti-glycolytic effects of resveratrol (RSV), a polyphenol known as a caloric restriction mimetic, and investigated its cytoprotective effects under glucose-deprived conditions in two-dimensional (2D) and 3D-cell culture systems using rat chondrocytes. In 2D culture, the low-glucose (LG, 0.5 mg/mL) condition caused time- and dose-dependent cell death in chondrocytes, whereas co-treatment with 50 μM RSV significantly restored cell viability under glucose deprivation. In RSV-treated cells, the expression levels of glycolytic genes (<em>GLUT1</em>, <em>PKM</em>, and <em>LDHA</em>) and glucose uptake were significantly downregulated, and phospho-AMPK levels were upregulated, indicating energy stress. RSV treatment restored the expression of extracellular matrix genes (<em>COL1A1</em> and <em>COL2A1</em>), which were downregulated under the LG condition, and augmented the pro-chondrogenic effect of TGF-β1 and ascorbic acid. In a 3D-culture model, spheroids constructed with RSV-pretreated chondrocytes had a more viable core region than dimethyl-sulfoxide-treated control spheroids. TGF-β-induced cartilage maturation led these spheroids to develop larger and more viable tissues than control spheroids. These results suggested that glycolytic inhibition by RSV decreased chondrocyte glucose usage, thereby preventing cell death caused by glucose deprivation. Our findings provide useful information for improving cell viability under hyponutrition conditions and can be applied to 3D tissue construction.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 2","pages":"Pages 90-97"},"PeriodicalIF":2.3,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lactic acid bacteria (LAB) play important roles in food microbiology and human health. Extracellular vesicles (EVs), which are drug transporters that contain functional components, derived from LAB have been recognized as materials with various beneficial effects, such as their anti-inflammatory effects. Owing to the complexity arising from strain-dependent functional differences in LAB, the functions of EVs derived from LAB have not been well elucidated. To investigate the strain-specific functions of EVs from LAB, we evaluated the ability of EVs from different LAB to induce interleukin 10 (IL-10) secretion by M2-like macrophages. The use of EVs with an increased capacity to induce IL-10 secretion and a G protein-coupled receptor (GPCR) assay revealed that EVs derived from specific lactic acid bacterial strains have agonistic effects against formyl peptide receptor 2 (FPR2). A strong and significant correlation between the ability of EVs derived from LAB to induce IL-10 secretion and agonistic activity against FPR2 was identified, and treatment with an FPR2 antagonist reduced the secretion of IL-10 induced by EVs from a specific lactic acid bacterial strain. Ultraviolet B (UVB) irradiation-induced damage to human keratinocytes was reversed after treatment with EVs with agonistic activity against FPR2, and this restorative effect was abolished by treatment with an FPR2 antagonist. In this study, we demonstrate for the first time that EVs derived from specific LAB have agonistic activity against FPR2; this activity could be a crucial factor in the anti-inflammatory effects of EVs released from specific LAB.
{"title":"Extracellular vesicles derived from specific lactic acid bacteria have agonistic activity against formyl peptide receptor 2","authors":"Hirosuke Sugahara, Keitaro Nagayama, Koichi Sashihara, Yasushi Nagatomi","doi":"10.1016/j.jbiosc.2025.04.009","DOIUrl":"10.1016/j.jbiosc.2025.04.009","url":null,"abstract":"<div><div>Lactic acid bacteria (LAB) play important roles in food microbiology and human health. Extracellular vesicles (EVs), which are drug transporters that contain functional components, derived from LAB have been recognized as materials with various beneficial effects, such as their anti-inflammatory effects. Owing to the complexity arising from strain-dependent functional differences in LAB, the functions of EVs derived from LAB have not been well elucidated. To investigate the strain-specific functions of EVs from LAB, we evaluated the ability of EVs from different LAB to induce interleukin 10 (IL-10) secretion by M2-like macrophages. The use of EVs with an increased capacity to induce IL-10 secretion and a G protein-coupled receptor (GPCR) assay revealed that EVs derived from specific lactic acid bacterial strains have agonistic effects against formyl peptide receptor 2 (FPR2). A strong and significant correlation between the ability of EVs derived from LAB to induce IL-10 secretion and agonistic activity against FPR2 was identified, and treatment with an FPR2 antagonist reduced the secretion of IL-10 induced by EVs from a specific lactic acid bacterial strain. Ultraviolet B (UVB) irradiation-induced damage to human keratinocytes was reversed after treatment with EVs with agonistic activity against FPR2, and this restorative effect was abolished by treatment with an FPR2 antagonist. In this study, we demonstrate for the first time that EVs derived from specific LAB have agonistic activity against FPR2; this activity could be a crucial factor in the anti-inflammatory effects of EVs released from specific LAB.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 21-27"},"PeriodicalIF":2.3,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144086247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-14DOI: 10.1016/j.jbiosc.2025.04.004
Huan Ren , Kazuaki Ninomiya
The objective of this study is to use the thermophilic acetogen Thermoanaerobacter kivui and a vitamin- and cysteine-free synthetic medium for an acetate-mediated two-stage microbial production of the biodegradable polymer poly[(R)-3-hydroxybutyrate] (PHB) from CO2 and H2. In the first stage, T. kivui could produce 10 g/L acetate within 14 days during anaerobic chemoautotrophic culture with substrate gas (H2:CO2 = 80:20) using cysteine-free DSMZ 171 medium (originally vitamin-free) in the presence of the pH neutralizer CaCO3. In the second stage, the acetate-containing spent medium obtained from the vitamin- and cysteine-free chemoautotrophic culture of T. kivui could be used for the aerobic culture of Cupriavidus necator for PHB production. Specifically, C. necator consumed all of the 5 g/L acetate in the spent medium of T. kivui for 36 h. The cell concentration of C. necator reached 1.2 g/L after 30 h of culture. The PHB content of C. necator reached 38 % (=0.46 g-PHB/L). The PHB yield was 0.11 g-PHB/g-acetate used. The present study can contribute to a sustainable production of bioplastic PHB from CO2 and H2 without vitamins and amino acids and without the risk of flammability problems caused by combustible gases (mixture of H2, O2, CO2).
{"title":"Acetate-mediated two-stage microbial production of poly[(R)-3-hydroxybutyrate] (PHB) from CO2 and H2 using a synthetic medium free of vitamins and cysteine","authors":"Huan Ren , Kazuaki Ninomiya","doi":"10.1016/j.jbiosc.2025.04.004","DOIUrl":"10.1016/j.jbiosc.2025.04.004","url":null,"abstract":"<div><div>The objective of this study is to use the thermophilic acetogen <em>Thermoanaerobacter kivui</em> and a vitamin- and cysteine-free synthetic medium for an acetate-mediated two-stage microbial production of the biodegradable polymer poly[(<em>R</em>)-3-hydroxybutyrate] (PHB) from CO<sub>2</sub> and H<sub>2</sub>. In the first stage, <em>T. kivui</em> could produce 10 g/L acetate within 14 days during anaerobic chemoautotrophic culture with substrate gas (H<sub>2</sub>:CO<sub>2</sub> = 80:20) using cysteine-free DSMZ 171 medium (originally vitamin-free) in the presence of the pH neutralizer CaCO<sub>3</sub>. In the second stage, the acetate-containing spent medium obtained from the vitamin- and cysteine-free chemoautotrophic culture of <em>T. kivui</em> could be used for the aerobic culture of <em>Cupriavidus necator</em> for PHB production. Specifically, <em>C. necator</em> consumed all of the 5 g/L acetate in the spent medium of <em>T. kivui</em> for 36 h. The cell concentration of <em>C. necator</em> reached 1.2 g/L after 30 h of culture. The PHB content of <em>C. necator</em> reached 38 % (=0.46 g-PHB/L). The PHB yield was 0.11 g-PHB/g-acetate used. The present study can contribute to a sustainable production of bioplastic PHB from CO<sub>2</sub> and H<sub>2</sub> without vitamins and amino acids and without the risk of flammability problems caused by combustible gases (mixture of H<sub>2</sub>, O<sub>2</sub>, CO<sub>2</sub>).</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 42-49"},"PeriodicalIF":2.3,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dried fruits have many advantages on condensed nutrients, food preservation, and availability through all seasons. This study investigated the longevity effects of five commercially available dried fruits (date, pineapple, fig, mango, and prune) on Caenorhabditis elegans. Each of the five dried fruits remarkably increased the lifespan compared with the control group. Especially, date fruit had the longest mean and maximum lifespan. In the analysis for the ratio of mean lifespan (MLS) to maximum lifespan in order to clarify their relationship, the ratio for date fruit was approximately 0.9, showing that it results in a longer MLS than other dried fruits. These results imply that date fruit consumption leads to a significantly greater increase in the MLS of C. elegans. The date fruit examined in this study did not contain syringic acid, a previously reported factor known to extend lifespan in date fruit, suggesting that other unidentified component(s) may contribute to its longevity-promoting effect.
{"title":"Date, a major dried fruit, extends the lifespan of Caenorhabditis elegans","authors":"Koji Kakugawa , Shion Uehara , Syunya Katoh , Naoki Arimatsu , Sho Ogaki , Koji Masumura , Hideki Murakawa , Kenji Arakawa , Masaki Mizunuma","doi":"10.1016/j.jbiosc.2025.04.006","DOIUrl":"10.1016/j.jbiosc.2025.04.006","url":null,"abstract":"<div><div>Dried fruits have many advantages on condensed nutrients, food preservation, and availability through all seasons. This study investigated the longevity effects of five commercially available dried fruits (date, pineapple, fig, mango, and prune) on <em>Caenorhabditis elegans</em>. Each of the five dried fruits remarkably increased the lifespan compared with the control group. Especially, date fruit had the longest mean and maximum lifespan. In the analysis for the ratio of mean lifespan (MLS) to maximum lifespan in order to clarify their relationship, the ratio for date fruit was approximately 0.9, showing that it results in a longer MLS than other dried fruits. These results imply that date fruit consumption leads to a significantly greater increase in the MLS of <em>C</em>. <em>elegans</em>. The date fruit examined in this study did not contain syringic acid, a previously reported factor known to extend lifespan in date fruit, suggesting that other unidentified component(s) may contribute to its longevity-promoting effect.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 36-41"},"PeriodicalIF":2.3,"publicationDate":"2025-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143998587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anaerobic conditions facilitate bioproduction by enabling diverse metabolic pathways; however, they disrupt redox balance due to the accumulation of reduced cofactors, limiting metabolic efficiency. Rhodoquinone (RQ), a low-redox-potential quinone, supports electron transport under anaerobic conditions. Unlike menaquinone, RQ is synthesized from ubiquinone through a single enzymatic reaction catalyzed by rhodoquinone biosynthesis protein A (RquA), making it a simple, adaptable metabolic engineering tool. In this study, RQ was synthesized in the menaquinone-deficient Escherichia coli ΔmenA strain via heterologous gene expression of rquA from Euglena gracilis. The engineered strain tripled succinate production under anaerobic conditions compared with the control strain. Redox analysis showed a decreased NADH/NAD+ ratio, reflecting improved electron flow under oxygen-limited conditions. Introducing rquA into a strain with high succinate production further increased succinate yields, confirming compatibility with existing metabolic modifications. To explore broader applications, rquA from Rhodospirillum rubrum was expressed in Cyanidioschyzon merolae mitochondria using a construct with a C. merolae mitochondrial targeting signal. Quinone analysis confirmed RQ synthesis, and the engineered strains produced more succinate anaerobically relative to the controls. Although redox cofactor ratios in C. merolae remained stable, rotenone sensitivity indicated altered mitochondrial electron transport under anaerobic conditions. These findings demonstrate that RQ synthesis enhances anaerobic metabolism in bacterial and eukaryotic systems, providing a versatile tool for metabolic engineering under oxygen-limited conditions.
{"title":"Engineering anaerobic electron flow through heterologous rhodoquinone synthesis in model microbial and photosynthetic platforms","authors":"Haruki Sasaki , Yuri Yanagihara , Yui Satofuka , Masaki Hasegawa , Yuichiro Kashiyama , Kimitoshi Sakamoto , Yoshihiro Toya , Hiroshi Shimizu , Takayuki Fujiwara , Shin'ya Miyagishima , Mitsuhiro Ueda , Tatsuji Sakamoto , Masami Nakazawa","doi":"10.1016/j.jbiosc.2025.04.008","DOIUrl":"10.1016/j.jbiosc.2025.04.008","url":null,"abstract":"<div><div>Anaerobic conditions facilitate bioproduction by enabling diverse metabolic pathways; however, they disrupt redox balance due to the accumulation of reduced cofactors, limiting metabolic efficiency. Rhodoquinone (RQ), a low-redox-potential quinone, supports electron transport under anaerobic conditions. Unlike menaquinone, RQ is synthesized from ubiquinone through a single enzymatic reaction catalyzed by rhodoquinone biosynthesis protein A (RquA), making it a simple, adaptable metabolic engineering tool. In this study, RQ was synthesized in the menaquinone-deficient <em>Escherichia coli</em> Δ<em>menA</em> strain via heterologous gene expression of <em>rquA</em> from <em>Euglena gracilis</em>. The engineered strain tripled succinate production under anaerobic conditions compared with the control strain. Redox analysis showed a decreased NADH/NAD<sup>+</sup> ratio, reflecting improved electron flow under oxygen-limited conditions. Introducing <em>rquA</em> into a strain with high succinate production further increased succinate yields, confirming compatibility with existing metabolic modifications. To explore broader applications, <em>rquA</em> from <em>Rhodospirillum rubrum</em> was expressed in <em>Cyanidioschyzon merolae</em> mitochondria using a construct with a <em>C. merolae</em> mitochondrial targeting signal. Quinone analysis confirmed RQ synthesis, and the engineered strains produced more succinate anaerobically relative to the controls. Although redox cofactor ratios in <em>C. merolae</em> remained stable, rotenone sensitivity indicated altered mitochondrial electron transport under anaerobic conditions. These findings demonstrate that RQ synthesis enhances anaerobic metabolism in bacterial and eukaryotic systems, providing a versatile tool for metabolic engineering under oxygen-limited conditions.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 14-20"},"PeriodicalIF":2.3,"publicationDate":"2025-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143965689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Japanese sake is fermented with specific strains of budding yeast Saccharomyces cerevisiae. Sake yeasts can allow the ethanol concentration of sake to exceed 20 % without distillation. While the genetic mutations responsible for these exceptional properties have been investigated, the underlying metabolism has not been fully explored. It is because yeast cells cultured in sake mash are difficult to collect for metabolome analysis. This study aimed to clarify the metabolic differences of K701 sake yeast and the X2180 diploid laboratory strain when cultured under sake fermentation conditions. To find an alternative medium that mimics sake fermentation and is applicable to measurements of intracellular metabolome, we compared three liquid media, including SD2 medium (synthetic dextrose medium containing 2 % glucose), SD20 medium (synthetic dextrose medium containing 20 % glucose and 1.8 % lactic acid) and pseudo-sake medium (a supernatant of saccharified rice supplemented with 1.8 % lactic acid). Culture profile data demonstrated that the pseudo-sake medium successfully reproduced the metabolic traits of K701 observed in sake mash. Targeted metabolome analysis of yeast cells cultured in the pseudo-sake medium revealed that levels of glycolytic metabolites, such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and fructose-1,6-bisphosphate (FBP), were significantly higher with K701. Based on metabolite concentration data, we inferred that K701 cells had a higher ATP regeneration rate. Calculation of differential Gibbs free energy changes revealed that the glucokinase reaction was upregulated in K701. The present study has, for the first time, revealed the metabolism of K701 sake yeast responsible for its exceptional fermentation ability under sake fermentation conditions.
{"title":"Comparative metabolome analysis of sake yeast with enhanced fermentation performance in sake fermentation conditions","authors":"Kotaro Mori , Taisuke Seike , Nobuyuki Okahashi , Toshinari Takahashi , Fumio Matsuda","doi":"10.1016/j.jbiosc.2025.04.001","DOIUrl":"10.1016/j.jbiosc.2025.04.001","url":null,"abstract":"<div><div>Japanese sake is fermented with specific strains of budding yeast <em>Saccharomyces cerevisiae</em>. Sake yeasts can allow the ethanol concentration of sake to exceed 20 % without distillation. While the genetic mutations responsible for these exceptional properties have been investigated, the underlying metabolism has not been fully explored. It is because yeast cells cultured in sake mash are difficult to collect for metabolome analysis. This study aimed to clarify the metabolic differences of K701 sake yeast and the X2180 diploid laboratory strain when cultured under sake fermentation conditions. To find an alternative medium that mimics sake fermentation and is applicable to measurements of intracellular metabolome, we compared three liquid media, including SD2 medium (synthetic dextrose medium containing 2 % glucose), SD20 medium (synthetic dextrose medium containing 20 % glucose and 1.8 % lactic acid) and pseudo-sake medium (a supernatant of saccharified rice supplemented with 1.8 % lactic acid). Culture profile data demonstrated that the pseudo-sake medium successfully reproduced the metabolic traits of K701 observed in sake mash. Targeted metabolome analysis of yeast cells cultured in the pseudo-sake medium revealed that levels of glycolytic metabolites, such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and fructose-1,6-bisphosphate (FBP), were significantly higher with K701. Based on metabolite concentration data, we inferred that K701 cells had a higher ATP regeneration rate. Calculation of differential Gibbs free energy changes revealed that the glucokinase reaction was upregulated in K701. The present study has, for the first time, revealed the metabolism of K701 sake yeast responsible for its exceptional fermentation ability under sake fermentation conditions.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 28-35"},"PeriodicalIF":2.3,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144019694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Streptomyces albulus NBRC14147 produces the polycationic homopoly(amino acid) ε-poly-l-lysine (ε-PL). Due to its antimicrobial properties, nontoxicity to humans, biodegradability, and permeability, there is a high demand for ε-PL. As ε-PL is produced by l-lysine polymerization, elucidating the source of l-lysine for ε-PL production is crucial for enhancing its yield. In actinobacteria, l-lysine is produced by diaminopimelate (DAP) pathway. In this study, 2,6-pyridine-dicarboxylate (PDC) was identified as the inhibitor of DapB, a DAP pathway enzyme, by comparing the structure of DapB from Mycobacterium tuberculosis with the model structure of DapB from S. albulus. We also found that adding PDC inhibited the growth of S. albulus. More importantly, PDC additions during the initial stages of the ε-PL production phase led to the accumulation of amino acids generated from pyruvate and l-aspartic 4-semialdehyde, while the ε-PL production was terminated. These findings suggest that de novo biosynthesized nascent l-lysine from the DAP pathway contributes to ε-PL production.
{"title":"Lysine source for ε-poly-l-lysine biosynthesis depends on diaminopimelate pathway during its production in Streptomyces albulus","authors":"Fumihito Hasebe , Daisuke Shimada , Chitose Maruyama , Yoshimitsu Hamano","doi":"10.1016/j.jbiosc.2025.04.005","DOIUrl":"10.1016/j.jbiosc.2025.04.005","url":null,"abstract":"<div><div><em>Streptomyces albulus</em> NBRC14147 produces the polycationic homopoly(amino acid) ε-poly-<span>l</span>-lysine (ε-PL). Due to its antimicrobial properties, nontoxicity to humans, biodegradability, and permeability, there is a high demand for ε-PL. As ε-PL is produced by <span>l</span>-lysine polymerization, elucidating the source of <span>l</span>-lysine for ε-PL production is crucial for enhancing its yield. In actinobacteria, <span>l</span>-lysine is produced by diaminopimelate (DAP) pathway. In this study, 2,6-pyridine-dicarboxylate (PDC) was identified as the inhibitor of DapB, a DAP pathway enzyme, by comparing the structure of DapB from <em>Mycobacterium tuberculosis</em> with the model structure of DapB from <em>S. albulus</em>. We also found that adding PDC inhibited the growth of <em>S. albulus</em>. More importantly, PDC additions during the initial stages of the ε-PL production phase led to the accumulation of amino acids generated from pyruvate and <span>l</span>-aspartic 4-semialdehyde, while the ε-PL production was terminated. These findings suggest that de novo biosynthesized nascent <span>l</span>-lysine from the DAP pathway contributes to ε-PL production.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"140 1","pages":"Pages 9-13"},"PeriodicalIF":2.3,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143993134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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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