Pub Date : 2025-12-20DOI: 10.1016/j.jbiosc.2025.11.008
Masashi Tsuda , Yuki Nakatani , Satoshi Baba , Kumi Yoshida , Kazuhiko Someya , Yoshikuni Onodera , Koichi Nonaka , Yasunori Chiba
The development of an effective vaccine against noroviruses remains a major public health priority. Norovirus GII.4 capsid protein VP1 as a promising vaccine candidate was produced by the methylotrophic yeast Ogataea minuta production system. It was intracellularly expressed and subsequently purified by immobilized metal affinity chromatography and anion exchange chromatography, yielding 13.4 mg of highly purified VP1 protein from 50 mL of culture supernatant. The formation of VP1-based virus-like particles (VLPs) that retained their structure even after freeze-thawing was confirmed by transmission electron microscopy. The VP1 protein purified in the form of VLPs displayed strong antigenicity and specific, dose-dependent binding to histo-blood group antigens, as determined by the enzyme-linked immunosorbent assay (ELISA). Immunogenicity studies in BALB/c mice demonstrated that intramuscular administration induced robust serum IgG responses across all tested doses, with no significant dose-dependent differences. Furthermore, mucosal administration intranasally or sublingually induced systemic IgG, systemic IgA, and mucosal IgA responses. These responses were significantly enhanced by the lipid A adjuvant. These findings showed that the O. minuta production system is capable of producing immunogenic Norovirus VLPs as a vaccine candidate.
{"title":"Production and characterization of norovirus virus-like particles vaccine candidates in a genetically modified Ogataea minuta system","authors":"Masashi Tsuda , Yuki Nakatani , Satoshi Baba , Kumi Yoshida , Kazuhiko Someya , Yoshikuni Onodera , Koichi Nonaka , Yasunori Chiba","doi":"10.1016/j.jbiosc.2025.11.008","DOIUrl":"10.1016/j.jbiosc.2025.11.008","url":null,"abstract":"<div><div>The development of an effective vaccine against noroviruses remains a major public health priority. Norovirus GII.4 capsid protein VP1 as a promising vaccine candidate was produced by the methylotrophic yeast <em>Ogataea minuta</em> production system. It was intracellularly expressed and subsequently purified by immobilized metal affinity chromatography and anion exchange chromatography, yielding 13.4 mg of highly purified VP1 protein from 50 mL of culture supernatant. The formation of VP1-based virus-like particles (VLPs) that retained their structure even after freeze-thawing was confirmed by transmission electron microscopy. The VP1 protein purified in the form of VLPs displayed strong antigenicity and specific, dose-dependent binding to histo-blood group antigens, as determined by the enzyme-linked immunosorbent assay (ELISA). Immunogenicity studies in BALB/c mice demonstrated that intramuscular administration induced robust serum IgG responses across all tested doses, with no significant dose-dependent differences. Furthermore, mucosal administration intranasally or sublingually induced systemic IgG, systemic IgA, and mucosal IgA responses. These responses were significantly enhanced by the lipid A adjuvant. These findings showed that the <em>O. minuta</em> production system is capable of producing immunogenic Norovirus VLPs as a vaccine candidate.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 165-171"},"PeriodicalIF":2.9,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804660","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}
Induced pluripotent stem cells (iPSCs) have significant potential for regenerative medicine, particularly for bone tissue engineering. While three-dimensional spheroid cultures enhance iPSC differentiation by better mimicking physiological conditions, spheroid size critically affects cell viability and differentiation ability. Microwell plates enable large-scale production of uniform spheroids and would be especially useful for regenerative medicine and tissue engineering. Here, we investigated the effect of spheroid size on the osteogenic differentiation of iPSCs using microwell plates to generate spheroids under the following conditions: Elp200 (microwell plate with 200/100 μm diameter/depth) and Elp900 (microwell plate with 900/700 μm). We observed that larger Elp900 spheroids promoted mesodermal differentiation more effectively, likely due to enhanced cell–cell interactions and altered internal microenvironments. However, Elp900 spheroids exhibited increased apoptosis in their core regions, evidenced by viability staining, transmission electron microscopy, and TUNEL staining. Upon dissociation and adherent culture, Elp900-derived cells demonstrated significantly higher expression of osteogenic markers (Runx2, Ibsp) and mineralization compared to Elp200-derived cells. Proteomic analysis revealed that apoptosis- and extracellular matrix (ECM)-related proteins, such as SERPINH1 and COL4A1, were upregulated in Elp900 cultures. These findings suggest that controlled apoptosis within larger spheroids may activate stress-related pathways, promote ECM formation, and enhance osteogenic differentiation by activating the TGF-β signaling pathway. Our findings highlight optimal spheroid sizing as a key factor for maximizing the efficiency and reproducibility of osteogenic differentiation of iPSCs, providing a foundation for improved strategies in iPSC-based bone tissue regeneration.
{"title":"Spheroid size-induced apoptosis enhances osteogenic differentiation of iPS cells","authors":"Hideto Tatsumi , Hiroko Okawa , Naruephorn Vinaikosol , Akihito Moribayashi , Hiroki Kayashima , Hirofumi Yatani , Hiroshi Egusa","doi":"10.1016/j.jbiosc.2025.11.010","DOIUrl":"10.1016/j.jbiosc.2025.11.010","url":null,"abstract":"<div><div>Induced pluripotent stem cells (iPSCs) have significant potential for regenerative medicine, particularly for bone tissue engineering. While three-dimensional spheroid cultures enhance iPSC differentiation by better mimicking physiological conditions, spheroid size critically affects cell viability and differentiation ability. Microwell plates enable large-scale production of uniform spheroids and would be especially useful for regenerative medicine and tissue engineering. Here, we investigated the effect of spheroid size on the osteogenic differentiation of iPSCs using microwell plates to generate spheroids under the following conditions: Elp200 (microwell plate with 200/100 μm diameter/depth) and Elp900 (microwell plate with 900/700 μm). We observed that larger Elp900 spheroids promoted mesodermal differentiation more effectively, likely due to enhanced cell–cell interactions and altered internal microenvironments. However, Elp900 spheroids exhibited increased apoptosis in their core regions, evidenced by viability staining, transmission electron microscopy, and TUNEL staining. Upon dissociation and adherent culture, Elp900-derived cells demonstrated significantly higher expression of osteogenic markers (<em>Runx2</em>, <em>Ibsp</em>) and mineralization compared to Elp200-derived cells. Proteomic analysis revealed that apoptosis- and extracellular matrix (ECM)-related proteins, such as SERPINH1 and COL4A1, were upregulated in Elp900 cultures. These findings suggest that controlled apoptosis within larger spheroids may activate stress-related pathways, promote ECM formation, and enhance osteogenic differentiation by activating the TGF-β signaling pathway. Our findings highlight optimal spheroid sizing as a key factor for maximizing the efficiency and reproducibility of osteogenic differentiation of iPSCs, providing a foundation for improved strategies in iPSC-based bone tissue regeneration.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 203-209"},"PeriodicalIF":2.9,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804722","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 : 2025-12-16DOI: 10.1016/j.jbiosc.2025.11.009
Rina Sakamaki , Takao Matsuba , Yasuyuki Kurihara
Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are membrane-bound vesicles secreted by cells. They play essential roles in intercellular communications and are involved in numerous physiological processes. Given their functional importance, EVs have emerged as promising tools for diagnosing and treating various diseases. In this study, we focused on the utility of EVs and explored their application in the analysis of contact-dependent cell–cell interactions, which are essential for the control of cell differentiation and induction of immune responses. Although several methods have been developed to evaluate these interactions, they often require complex procedures and advanced optimization, limiting their broad applicability. To overcome these limitations, we developed a novel method utilizing EVs to present membrane proteins in their native conformations. Our strategy involved producing fluorescently labeled EVs with target antigens and quantitatively assessing their binding to target cells via flow cytometry. Using fluorescently labeled EVs presenting with either an N-terminal pro-brain natriuretic peptide or interleukin-2 receptor, we successfully detected specific interactions with corresponding hybridoma B cell receptors. This simpler method requires no advanced optimization and effectively analyzes cell–cell interactions under physiological conditions in a high-throughput and quantitative manner. Our findings highlight the potential of this EV-based system as a valuable tool for studying membrane protein-mediated cell–cell interactions in bioscience research.
{"title":"Establishment of a simple and novel method for contact-dependent intercellular interaction analysis using extracellular vesicles","authors":"Rina Sakamaki , Takao Matsuba , Yasuyuki Kurihara","doi":"10.1016/j.jbiosc.2025.11.009","DOIUrl":"10.1016/j.jbiosc.2025.11.009","url":null,"abstract":"<div><div>Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are membrane-bound vesicles secreted by cells. They play essential roles in intercellular communications and are involved in numerous physiological processes. Given their functional importance, EVs have emerged as promising tools for diagnosing and treating various diseases. In this study, we focused on the utility of EVs and explored their application in the analysis of contact-dependent cell–cell interactions, which are essential for the control of cell differentiation and induction of immune responses. Although several methods have been developed to evaluate these interactions, they often require complex procedures and advanced optimization, limiting their broad applicability. To overcome these limitations, we developed a novel method utilizing EVs to present membrane proteins in their native conformations. Our strategy involved producing fluorescently labeled EVs with target antigens and quantitatively assessing their binding to target cells via flow cytometry. Using fluorescently labeled EVs presenting with either an N-terminal pro-brain natriuretic peptide or interleukin-2 receptor, we successfully detected specific interactions with corresponding hybridoma B cell receptors. This simpler method requires no advanced optimization and effectively analyzes cell–cell interactions under physiological conditions in a high-throughput and quantitative manner. Our findings highlight the potential of this EV-based system as a valuable tool for studying membrane protein-mediated cell–cell interactions in bioscience research.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 194-202"},"PeriodicalIF":2.9,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145774699","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}
Synthetic biology approaches enable the creation of promising chassis for practical application in various fields, though engineering of microbial metabolism often imposes a metabolic burden, potentially driving adaptive evolution during long-term cultivation. A previously established phosphite (Pt)-dependent metabolic system has proven to be an effective strategy for the containment of genetically engineered microorganisms, although its implementation accompanied a slight growth retardation. Here, we investigated the effect of long-term serial passaging cultivation on the Pt-dependent strain of Synechococcus elongatus PCC 7942, RH714. Compared with the originally constructed RH714, the passaged population of RH714 exhibited improved growth and a higher rate of Pt consumption in culture medium. Sequence analysis revealed point mutations within the introduced htxBCDE transporter genes, which are required for selective incorporation of Pt as a phosphorus nutrition. Introduction of the mutated gene cluster into S. elongatus PCC 7942 reproduced the traits of the passaged RH714 population, suggesting that these genetic changes enhance Pt transport activity and account for the observed phenotypes. Disruption of endogenous phosphate (Pi) transporter genes in the strains expressing the mutated htxBCDE-ptxD cluster abolished growth in Pi-containing medium, suggesting that the mutations in the transporter genes did not alter substrate specificity toward Pi. These results indicated that long-term passage cultivation developed an optimized mutant capable of efficient proliferation under the Pt metabolizing conditions without compromising its biocontainment capability.
{"title":"Adaptive laboratory evolution optimizes an engineered phosphite utilization pathway in Synechococcus elongatus PCC 7942","authors":"Hiroki Murakami , Naoki Momokawa , Kei Motomura , Akio Kuroda , Ryuichi Hirota","doi":"10.1016/j.jbiosc.2025.11.006","DOIUrl":"10.1016/j.jbiosc.2025.11.006","url":null,"abstract":"<div><div>Synthetic biology approaches enable the creation of promising chassis for practical application in various fields, though engineering of microbial metabolism often imposes a metabolic burden, potentially driving adaptive evolution during long-term cultivation. A previously established phosphite (Pt)-dependent metabolic system has proven to be an effective strategy for the containment of genetically engineered microorganisms, although its implementation accompanied a slight growth retardation. Here, we investigated the effect of long-term serial passaging cultivation on the Pt-dependent strain of <em>Synechococcus elongatus</em> PCC 7942, RH714. Compared with the originally constructed RH714, the passaged population of RH714 exhibited improved growth and a higher rate of Pt consumption in culture medium. Sequence analysis revealed point mutations within the introduced <em>htxBCDE</em> transporter genes, which are required for selective incorporation of Pt as a phosphorus nutrition. Introduction of the mutated gene cluster into <em>S. elongatus</em> PCC 7942 reproduced the traits of the passaged RH714 population, suggesting that these genetic changes enhance Pt transport activity and account for the observed phenotypes. Disruption of endogenous phosphate (Pi) transporter genes in the strains expressing the mutated <em>htxBCDE-ptxD</em> cluster abolished growth in Pi-containing medium, suggesting that the mutations in the transporter genes did not alter substrate specificity toward Pi. These results indicated that long-term passage cultivation developed an optimized mutant capable of efficient proliferation under the Pt metabolizing conditions without compromising its biocontainment capability.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 152-157"},"PeriodicalIF":2.9,"publicationDate":"2025-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145751920","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}
Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating and shaping adaptive immune responses. Targeting antigens to DCs has emerged as a promising strategy to enhance vaccine efficacy and tailor desired immune responses. While antibodies are well established as targeting molecules for DCs, the use of peptides remains underexplored despite their favorable tissue penetration and their ability to offer design flexibility. Here, we report the identification of dectin-1-binding peptides using a ribosome display-based in vitro directed evolution system. Dectin-1 is a C-type lectin receptor expressed on murine CD11b+ and human CD1c+ DCs, which plays a key role in antigen uptake and in directing immune responses toward specific pathways, particularly the Th17 pathway. Using recombinant murine dectin-1 as bait, we performed four rounds of ribosome display selection and obtained peptides with high affinity. Selected peptides fused to enhanced green fluorescent protein showed binding to recombinant dectin-1 and native dectin-1-expressing cells, including bone marrow-derived DCs. These results demonstrated the feasibility of peptide-based molecular targeting toward dectin-1+ DCs, which would not only provide a versatile platform for the development of next-generation vaccines and immunotherapies, but also a valuable tool for dissecting the functional roles of dectin-1+ DCs in immune regulation.
{"title":"Development of dectin-1-binding peptides targeting dendritic cells for antigen delivery via ribosome display","authors":"Yoshirou Kawaguchi , Md Shahin Sarker , Mina Yokoyama , Misuzu Nakaya , Takanatsu Hosokawa , Noriho Kamiya , Masahiro Goto","doi":"10.1016/j.jbiosc.2025.11.005","DOIUrl":"10.1016/j.jbiosc.2025.11.005","url":null,"abstract":"<div><div>Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating and shaping adaptive immune responses. Targeting antigens to DCs has emerged as a promising strategy to enhance vaccine efficacy and tailor desired immune responses. While antibodies are well established as targeting molecules for DCs, the use of peptides remains underexplored despite their favorable tissue penetration and their ability to offer design flexibility. Here, we report the identification of dectin-1-binding peptides using a ribosome display-based in vitro directed evolution system. Dectin-1 is a C-type lectin receptor expressed on murine CD11b<sup>+</sup> and human CD1c<sup>+</sup> DCs, which plays a key role in antigen uptake and in directing immune responses toward specific pathways, particularly the Th17 pathway. Using recombinant murine dectin-1 as bait, we performed four rounds of ribosome display selection and obtained peptides with high affinity. Selected peptides fused to enhanced green fluorescent protein showed binding to recombinant dectin-1 and native dectin-1-expressing cells, including bone marrow-derived DCs. These results demonstrated the feasibility of peptide-based molecular targeting toward dectin-1<sup>+</sup> DCs, which would not only provide a versatile platform for the development of next-generation vaccines and immunotherapies, but also a valuable tool for dissecting the functional roles of dectin-1<sup>+</sup> DCs in immune regulation.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 158-164"},"PeriodicalIF":2.9,"publicationDate":"2025-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145701096","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 : 2025-12-05DOI: 10.1016/j.jbiosc.2025.11.004
Nevin McCone , Masahito Hosokawa
Single-cell genomics (SCG) complements culture-independent metagenomics for accessing fungal genomes, particularly from lineages that remain uncultured. We contrast metagenomics, which excels when profiling community composition and metabolic potential but often underrepresents low-abundance fungi, with SCG, which first isolates individual cells or nuclei to generate single-amplified genomes (SAGs) and can recover rare or microdiverse taxa. We then organize existing fungal SCG applications into three subgroups: spore-level sequencing from host-enriched or environmental material; single-nucleus genomics for multinucleate fungi; and single-spore sequencing of haploid progeny for diploid linkage and chromosome phasing. Across studies, pooling and co-assembly of cognate cells improves completeness; key hurdles persist in wall lysis, whole-genome amplification bias, and contamination control. Practical advances include shallow sequencing for QC triage, nuclei pooling with normalized co-assembly, and hybrid long- and short-read assembly. SCG adds unique value where strain resolution and genotypic context matter, including host-to-mobile-element linkage, recovery of large biosynthetic gene clusters, and karyotype validation against telomere-to-telomere references. Used alongside metagenomics, SCG enables a strain-resolved view of fungal biodiversity and function, with incremental improvements across the SCG pipeline promising routine access to genomes from early-diverging and other environmentally embedded fungi.
{"title":"Recovering genomes from uncultured fungi with single-cell genomics","authors":"Nevin McCone , Masahito Hosokawa","doi":"10.1016/j.jbiosc.2025.11.004","DOIUrl":"10.1016/j.jbiosc.2025.11.004","url":null,"abstract":"<div><div>Single-cell genomics (SCG) complements culture-independent metagenomics for accessing fungal genomes, particularly from lineages that remain uncultured. We contrast metagenomics, which excels when profiling community composition and metabolic potential but often underrepresents low-abundance fungi, with SCG, which first isolates individual cells or nuclei to generate single-amplified genomes (SAGs) and can recover rare or microdiverse taxa. We then organize existing fungal SCG applications into three subgroups: spore-level sequencing from host-enriched or environmental material; single-nucleus genomics for multinucleate fungi; and single-spore sequencing of haploid progeny for diploid linkage and chromosome phasing. Across studies, pooling and co-assembly of cognate cells improves completeness; key hurdles persist in wall lysis, whole-genome amplification bias, and contamination control. Practical advances include shallow sequencing for QC triage, nuclei pooling with normalized co-assembly, and hybrid long- and short-read assembly. SCG adds unique value where strain resolution and genotypic context matter, including host-to-mobile-element linkage, recovery of large biosynthetic gene clusters, and karyotype validation against telomere-to-telomere references. Used alongside metagenomics, SCG enables a strain-resolved view of fungal biodiversity and function, with incremental improvements across the SCG pipeline promising routine access to genomes from early-diverging and other environmentally embedded fungi.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 143-151"},"PeriodicalIF":2.9,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145695859","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}
Microcarrier surface topography and fluid shear stress (FSS) critically regulate cellular behavior. An edible zein/fucoidan microcarrier with 200 μm grooves was designed for this study. Computational fluid dynamics (CFD) simulations and experiments were combined to analyze surface microfluidic characteristics during dynamic culture and their cellular effects. The research demonstrates controlled myogenic differentiation through groove topography and FSS modulation for scalable cultured meat production. The results demonstrated that the grooved microcarriers exhibited excellent cell attachment and proliferation capacity in spinner flask dynamic culture, achieving a maximum cell density of 1.16 × 106 cells/mL, comparable to commercial Cultispher-S microcarriers. The groove structure promoted cell alignment through contact guidance, significantly enhancing the gene expression of myogenic differentiation markers (myogenic differentiation 1, MyoD1; α-actinin; myosin heavy chain, MHC) and cell fusion (myomaker, MYMK). CFD simulations revealed that the grooves created a low-shear microenvironment (minimum average FSS: 3.93 × 10−2 Pa, maximum: 1.18 × 10−1 Pa), which effectively avoided high FSS-induced damage while maintaining mechanical stimulation. This optimal mechanical microenvironment further activated the expression of key genes involved in early-stage (MyoD1; myogenin, MyoG; myocyte enhancer factor 2C, MEF2C) and late-stage (α-actinin; myosin heavy chain 2, Myh2) myogenic differentiation. Flat and spherical microcarriers showed lower myogenic differentiation efficiency. This study elucidates the synergistic mechanism between groove structures and the low FSS microenvironment within grooves, providing a novel scaffold design rationale that combines biomimetic topology with fluid dynamics compatibility for large-scale cultured meat production.
{"title":"Zein/Fucoidan microcarriers promote myogenic differentiation via topographical cues and hydrodynamic modulation","authors":"Wanli Xiong, Chengxin Ge, Botao Zhang, Ziying Chen, Yuzhe Guo, Qiaohui Lu, Wen-song Tan, Yan Zhou","doi":"10.1016/j.jbiosc.2025.11.003","DOIUrl":"10.1016/j.jbiosc.2025.11.003","url":null,"abstract":"<div><div>Microcarrier surface topography and fluid shear stress (FSS) critically regulate cellular behavior. An edible zein/fucoidan microcarrier with 200 μm grooves was designed for this study. Computational fluid dynamics (CFD) simulations and experiments were combined to analyze surface microfluidic characteristics during dynamic culture and their cellular effects. The research demonstrates controlled myogenic differentiation through groove topography and FSS modulation for scalable cultured meat production. The results demonstrated that the grooved microcarriers exhibited excellent cell attachment and proliferation capacity in spinner flask dynamic culture, achieving a maximum cell density of 1.16 × 10<sup>6</sup> cells/mL, comparable to commercial Cultispher-S microcarriers. The groove structure promoted cell alignment through contact guidance, significantly enhancing the gene expression of myogenic differentiation markers (myogenic differentiation 1, MyoD1; α-actinin; myosin heavy chain, MHC) and cell fusion (myomaker, MYMK). CFD simulations revealed that the grooves created a low-shear microenvironment (minimum average FSS: 3.93 × 10<sup>−2</sup> Pa, maximum: 1.18 × 10<sup>−1</sup> Pa), which effectively avoided high FSS-induced damage while maintaining mechanical stimulation. This optimal mechanical microenvironment further activated the expression of key genes involved in early-stage (MyoD1; myogenin, MyoG; myocyte enhancer factor 2C, MEF2C) and late-stage (α-actinin; myosin heavy chain 2, Myh2) myogenic differentiation. Flat and spherical microcarriers showed lower myogenic differentiation efficiency. This study elucidates the synergistic mechanism between groove structures and the low FSS microenvironment within grooves, providing a novel scaffold design rationale that combines biomimetic topology with fluid dynamics compatibility for large-scale cultured meat production.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 3","pages":"Pages 172-184"},"PeriodicalIF":2.9,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145687478","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}
The natural abundances of stable isotopes were used to determine prey–predator relationships and material flows in a biological treatment reactor for municipal wastewater. The reactor used in this study was a down-flow hanging sponge (DHS), which is an alternative trickling filter that uses sponge as the packing material. The sponge retained sludge containing a wide variety of organisms, including microfauna. Stable isotope analysis revealed spatial, temporal, and biotic variations in the carbon and nitrogen stable isotope ratios (δ13C and δ15N) of the retained sludge and microfauna (water mites and fly larvae). In addition, adult flies and spiders were present and analyzed. The δ13C and δ15N in sludge were temporally and spatially similar along the reactor. The isotopic signature was associated with treatment characteristics such as a low nitrification efficiency in the DHS reactor. The δ13C and δ15N of sympatric fly larvae differed from those of water mites, which indicated dietary differences between the taxa. Interestingly, the water mites had higher δ13C and δ15N than the retained sludge, which indicated that they were in a higher trophic position in the food web. In addition, the δ13C and δ15N values of spiders were approximately 1 ‰–3‰ higher than those of adult flies. This strongly suggested that a prey–predator relationship existed between adult flies and spiders.
{"title":"Spatial, temporal, and biotic changes in the natural abundances of stable carbon and nitrogen isotopes in a biological treatment reactor","authors":"Takashi Onodera , Kengo Kubota , Akinori Iguchi , Akihiro Nagamachi , Tadashi Tagawa , Gen Kanaya , Ayato Kohzu , Kazuaki Syutsubo","doi":"10.1016/j.jbiosc.2025.11.002","DOIUrl":"10.1016/j.jbiosc.2025.11.002","url":null,"abstract":"<div><div>The natural abundances of stable isotopes were used to determine prey–predator relationships and material flows in a biological treatment reactor for municipal wastewater. The reactor used in this study was a down-flow hanging sponge (DHS), which is an alternative trickling filter that uses sponge as the packing material. The sponge retained sludge containing a wide variety of organisms, including microfauna. Stable isotope analysis revealed spatial, temporal, and biotic variations in the carbon and nitrogen stable isotope ratios (<em>δ</em><sup>13</sup>C and <em>δ</em><sup>15</sup>N) of the retained sludge and microfauna (water mites and fly larvae). In addition, adult flies and spiders were present and analyzed. The <em>δ</em><sup>13</sup>C and <em>δ</em><sup>15</sup>N in sludge were temporally and spatially similar along the reactor. The isotopic signature was associated with treatment characteristics such as a low nitrification efficiency in the DHS reactor. The <em>δ</em><sup>13</sup>C and <em>δ</em><sup>15</sup>N of sympatric fly larvae differed from those of water mites, which indicated dietary differences between the taxa. Interestingly, the water mites had higher <em>δ</em><sup>13</sup>C and <em>δ</em><sup>15</sup>N than the retained sludge, which indicated that they were in a higher trophic position in the food web. In addition, the <em>δ</em><sup>13</sup>C and <em>δ</em><sup>15</sup>N values of spiders were approximately 1 ‰–3‰ higher than those of adult flies. This strongly suggested that a prey–predator relationship existed between adult flies and spiders.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 2","pages":"Pages 135-141"},"PeriodicalIF":2.9,"publicationDate":"2025-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145633959","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}
A high growth rate is essential for increasing protein production efficiency in liquid fermentation of filamentous fungi, such as Aspergillus oryzae. However, the increase in culture viscosity due to fungal growth constrains the overall yield. We have demonstrated that culture viscosity is lower in A. oryzae AGΔ-GAGΔ strains, which are deficient in the cell surface polysaccharides α-1,3-glucan (AG) and galactosaminogalactan (GAG), than in the wild-type (WT) strains. Nevertheless, even in aerated fermentation, an increase in AGΔ-GAGΔ viscosity results in oxygen depletion, which limits fungal growth and enzyme production. In this study, we investigated viscosity dynamics and protein production during high-cell-density fermentation of AGΔ-GAGΔ under pure oxygen aeration. Fed-batch cultivation of the WT and AGΔ-GAGΔ strains, expressing recombinant xylanase (XynF1), was used to compare the effects of air and pure oxygen aeration at the same flow rate. At 60 h, AGΔ-GAGΔ under pure oxygen aeration showed higher cell density (1.2× WT under pure oxygen aeration, 2.1× AGΔ-GAGΔ under air aeration) and XynF1 activity (1.8× WT under pure oxygen aeration, 2.3× AGΔ-GAGΔ under air aeration). Under pure oxygen aeration, AGΔ-GAGΔ showed lower viscosity (0.32×) and mixing time (0.50×) than WT. At 60 h, fine mycelial pellets (micropellets; 200–700 μm) were clearly observed in AGΔ-GAGΔ under pure oxygen but not under air aeration. These findings suggest that oxygen enrichment during AGΔ-GAGΔ cultivation mitigated the increase in viscosity, thereby promoting higher cell density and protein production. The formation of micropellets in AGΔ-GAGΔ likely contributed to a reduction in culture viscosity.
{"title":"Using pure oxygen aeration to increase recombinant protein production by an Aspergillus oryzae hyphal dispersion strain","authors":"Satoshi Ishibashi , Shunya Susukida , Kiyoaki Muto , Ken Miyazawa , Silai Zhang , Akira Yoshimi , Eiji Tabata , Keietsu Abe","doi":"10.1016/j.jbiosc.2025.10.011","DOIUrl":"10.1016/j.jbiosc.2025.10.011","url":null,"abstract":"<div><div>A high growth rate is essential for increasing protein production efficiency in liquid fermentation of filamentous fungi, such as <em>Aspergillus oryzae</em>. However, the increase in culture viscosity due to fungal growth constrains the overall yield. We have demonstrated that culture viscosity is lower in <em>A. oryzae</em> AGΔ-GAGΔ strains, which are deficient in the cell surface polysaccharides α-1,3-glucan (AG) and galactosaminogalactan (GAG), than in the wild-type (WT) strains. Nevertheless, even in aerated fermentation, an increase in AGΔ-GAGΔ viscosity results in oxygen depletion, which limits fungal growth and enzyme production. In this study, we investigated viscosity dynamics and protein production during high-cell-density fermentation of AGΔ-GAGΔ under pure oxygen aeration. Fed-batch cultivation of the WT and AGΔ-GAGΔ strains, expressing recombinant xylanase (XynF1), was used to compare the effects of air and pure oxygen aeration at the same flow rate. At 60 h, AGΔ-GAGΔ under pure oxygen aeration showed higher cell density (1.2× WT under pure oxygen aeration, 2.1× AGΔ-GAGΔ under air aeration) and XynF1 activity (1.8× WT under pure oxygen aeration, 2.3× AGΔ-GAGΔ under air aeration). Under pure oxygen aeration, AGΔ-GAGΔ showed lower viscosity (0.32×) and mixing time (0.50×) than WT. At 60 h, fine mycelial pellets (micropellets; 200–700 μm) were clearly observed in AGΔ-GAGΔ under pure oxygen but not under air aeration. These findings suggest that oxygen enrichment during AGΔ-GAGΔ cultivation mitigated the increase in viscosity, thereby promoting higher cell density and protein production. The formation of micropellets in AGΔ-GAGΔ likely contributed to a reduction in culture viscosity.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 2","pages":"Pages 98-107"},"PeriodicalIF":2.9,"publicationDate":"2025-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145604529","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}
In this study, Pseudomonas sp. NGC7 was engineered to selectively produce vanillate (VA) from aromatic compounds in the sugarcane bagasse alkaline extract. A VA O-demethylase gene (vanA4B4)-disrupted strain derived from NGC7 grew and produced VA from the extract containing no saccharides. The organic acids in the extract promoted the strain to grow. The aromatics in the extract were further concentrated by the solid phase extraction with DIAION HP20 resin, and the organic acids were fractionated into the flow-through fraction. A fed-batch culture of NGC7ΔvanA4B4 strain using this concentrated alkaline extract exhibited increased VA production; however, the accumulation of syringate (SA) and 4-hydroxybenzoate (HBA) was also observed along with VA production, despite the strain possessing the genes responsible for SA and HBA degradation. Analysis of the mutants capable of degrading SA while producing VA revealed that mutations in vanR2, a transcriptional repressor of the genes responsible for SA degradation, enabled SA degradation during VA production. In addition, the expression of praI, an HBA hydroxylase derived from Paenibacillus sp. JJ-1b, was suitable for efficient HBA degradation. Thus, the mutation in vanR2 and the expression of praI represented the key engineering strategies for achieving the selective VA production. As the growth of the engineered strains was promoted by the organic acids present in the extract, VA production from the concentrated extract was evaluated in a flow-through-based medium supplemented with mineral salts and metals. Finally, the engineered VA-producing strain produced 4.30 mM VA selectively at a yield of 77 mol% in the practical medium.
本研究对假单胞菌sp. NGC7进行了工程改造，使其能够从甘蔗渣碱性提取物中的芳香化合物中选择性地产生香草酸（VA）。从NGC7衍生的VA o -去甲基化酶基因（vanA4B4）中断菌株生长并从不含糖的提取物中产生VA。提取物中的有机酸促进了菌株的生长。用DIAION HP20树脂固相萃取进一步浓缩提取液中的芳烃，将有机酸分馏成流动馏分。使用该浓缩碱性提取物对NGC7ΔvanA4B4菌株进行分批补料培养，VA产量增加；然而，尽管菌株具有负责SA和HBA降解的基因，但在产生VA的同时，也观察到紫丁香酸（SA）和4-羟基苯甲酸（HBA）的积累。对能够降解SA同时产生VA的突变体的分析表明，vanR2（负责SA降解的基因的转录抑制因子）的突变使SA在VA产生过程中降解。此外，源自Paenibacillus sp. JJ-1b的HBA羟化酶praI的表达适合于HBA的高效降解。因此，vanR2的突变和praI的表达代表了实现选择性VA产生的关键工程策略。由于提取物中存在的有机酸促进了工程菌株的生长，因此在添加无机盐和金属的流动培养基中评估了浓缩提取物的VA产量。最后，该工程产VA菌株在实际培养基中选择性地产生4.30 mM VA，产率为77 mol%。
{"title":"Selective vanillate production from sugarcane bagasse-derived aromatic compounds using an engineered Pseudomonas sp. NGC7-based strain","authors":"Md. Jahangir Alam , Naoya Kodama , Kazuma Ikeda , Kanami Muraki , Yudai Higuchi , Masaya Fujita , Naofumi Kamimura , Eiji Masai , Hiroyuki Kurihara , Tomonori Sonoki","doi":"10.1016/j.jbiosc.2025.11.001","DOIUrl":"10.1016/j.jbiosc.2025.11.001","url":null,"abstract":"<div><div>In this study, <em>Pseudomonas</em> sp. NGC7 was engineered to selectively produce vanillate (VA) from aromatic compounds in the sugarcane bagasse alkaline extract. A VA <em>O</em>-demethylase gene (<em>vanA4B4</em>)-disrupted strain derived from NGC7 grew and produced VA from the extract containing no saccharides. The organic acids in the extract promoted the strain to grow. The aromatics in the extract were further concentrated by the solid phase extraction with DIAION HP20 resin, and the organic acids were fractionated into the flow-through fraction. A fed-batch culture of NGC7Δ<em>vanA4B4</em> strain using this concentrated alkaline extract exhibited increased VA production; however, the accumulation of syringate (SA) and 4-hydroxybenzoate (HBA) was also observed along with VA production, despite the strain possessing the genes responsible for SA and HBA degradation. Analysis of the mutants capable of degrading SA while producing VA revealed that mutations in <em>vanR2</em>, a transcriptional repressor of the genes responsible for SA degradation, enabled SA degradation during VA production. In addition, the expression of <em>praI</em>, an HBA hydroxylase derived from <em>Paenibacillus</em> sp. JJ-1b, was suitable for efficient HBA degradation. Thus, the mutation in <em>vanR2</em> and the expression of <em>praI</em> represented the key engineering strategies for achieving the selective VA production. As the growth of the engineered strains was promoted by the organic acids present in the extract, VA production from the concentrated extract was evaluated in a flow-through-based medium supplemented with mineral salts and metals. Finally, the engineered VA-producing strain produced 4.30 mM VA selectively at a yield of 77 mol% in the practical medium.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"141 2","pages":"Pages 125-134"},"PeriodicalIF":2.9,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145581946","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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