Journal of bioscience and bioengineering最新文献

White-rot basidiomycete fungi have been the focus of research as edible mushrooms and for industrial utilization, especially due to their unique wood-degrading enzymes. Nevertheless, establishing highly efficient transformation systems for these organisms remains a substantial challenge. In filamentous ascomycetes, plasmids carrying the specific sequence, AMA1, have been widely used to enhance transformation efficiency. However, their functionality in basidiomycetes has not been systematically assessed. In this study, a pAMAHyg plasmid was constructed that harbored AMA1, in addition to a hygromycin B resistance marker gene derived from the pPHT1 plasmid. The pAMAHyg was introduced into the white-rot fungus Pleurotus ostreatus and yielded more hygromycin-resistant colonies than those with the pPHT1, despite its larger size, suggesting a positive impact on transformation efficiency. However, unlike previous reports in ascomycetes, frequent loss of drug resistance was not observed upon subculturing. Plasmid rescue experiments in Escherichia coli suggested that the circular form and original sequence of pPHT1 or pAMAHyg could be maintained in P. ostreatus, with few exceptions. Methylation-sensitive restriction enzyme analysis of the rescued plasmids confirmed the loss of bacterial Dam methylation and the acquisition of CpG methylation, regardless of AMA1 presence, suggesting the replication of these plasmids in P. ostreatus. Southern blot analysis revealed the genomic integration of the introduced plasmid as well. Collectively, these results suggest that the integrated and episomal plasmid sequences may coexist in P. ostreatus transformants. This study provides experimental evidence supporting the extrachromosomal existence of circular plasmids in the fungus, even in the absence of AMA1.

Bark compost serves as an organic fertilizer, originating from the microbial decomposition of tree bark and woody residues. An examination of 84 bacterial isolates from commercially available bark composts identified nine strains capable of degrading N-acylhomoserine lactones (AHLs), which function as signaling molecules in bacterial quorum sensing. Whole-genome sequencing of one AHL-degrading isolate, Ochrobactrum quorumnocens B44, revealed the presence of multiple AHL-degrading gene homologs, with the gene OCHQB44_21820 (aiiO) encoding the principal AHL-degrading enzyme. AiiO efficiently degraded a range of AHL molecules with acyl chain lengths of 6-10, including their 3-oxo derivatives. Contrary to previous reports suggesting that AiiO functions as an AHL acylase, hydrolyzing the amide bond of AHL, biochemical analysis in this study demonstrated that AiiO acts as an AHL lactonase, catalyzing the hydrolytic opening of the lactone ring in AHL. The purified AiiO as a maltose binding protein fusion efficiently degraded AHLs at an optimal temperature of 40 °C, maintaining stability under typical soil conditions. The expression of aiiO in the plant pathogen Pectobacterium carotovorum subsp. carotovorum causes self-degradation of biosynthesized AHL and suppresses potato tissue maceration regulated by AHL-mediated quorum sensing. Co-inoculation of B44 with P. carotovorum subsp. carotovorum attenuated soft rot symptoms in potato slices. These results showed the possibility that the application of bark compost rich in AHL-degrading bacteria enhances soil resistance to bacterial diseases.

Monoclonal antibodies (mAbs) are essential tools in life sciences and medicine because they provide high specificity and selective binding properties. However, their production is labor-intensive. Conventional hybridoma screening using the limiting dilution method requires repeated manual handling and long-term culture, which slows the isolation of target antibodies. To facilitate this process, we previously developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method that directly detects interactions between membrane-bound B-cell receptors and fluorescently labeled antigens. However, the preparation of multiple soluble antigens remains a challenge. Hence, we designed a single recombinant antigen, X-His-BAP-MBP (X-HBM), in which the target antigen (X) was fused to a solubilization tag (MBP), biotinylation tag (BAP), and His-tag for purification. We established a dual-color MIHS system that integrates immunization, screening, and evaluation into a workflow. Dual-color fluorescence labeling enabled the discrimination and exclusion of hybridomas producing antibodies against MBP or tag sequences, allowing the precise isolation of hybridomas secreting antibodies specific to the target antigen. Using the mitochondrial enzyme CARS2 as a model antigen, the proportion of target-positive hybridomas increased approximately eightfold compared to that using the conventional method. Specific mAbs were successfully obtained using only a single soluble antigen preparation through the MIHS method. This method supports the efficient selection of antigen-specific hybridomas, even when solubilization tags are used, by achieving both antigen specificity and practical solubility.

Single-cell arrays are essential tools for visualizing cellular heterogeneity and for linking cellular phenotypes observed by imaging to intracellular molecular systems through molecular analyses of cells retrieved after imaging. However, conventional single-cell array techniques often suffer from limited applicability due to the narrow microstructures required for single-cell trapping. In this study, we developed a dual-photoresponsive surface that enables single-cell photopatterning without any microstructures, as well as the selective light-induced release of individual cells of interest. The surface is based on a photoclickable material coating, onto which a photocleavable cell-anchoring material is photopatterned for single-cell trapping. We demonstrated light-induced patterning of mammalian immune cells through binding to patterned cell-anchoring materials, enabling microstructure-free single-cell array formation on a flat glass substrate. Furthermore, photopatterned individual cells were selectively released via localized light irradiation and subsequently retrieved at the single-cell level using cell pickers. These results represent the first demonstration of selective cell isolation from photoclickable surfaces following cell patterning and phenotypic observation. This surface is expected to be broadly applicable to a wide range of single-cell analyses by freely tailoring cell-anchoring molecules, designed cell patterns, and downstream molecular analyses.

Multi-organ microphysiological systems (multi-organ MPSs) are in vitro platforms that simulate interactions between multiple organs in the body. However, most existing multi-organ MPSs often suffer from high complexity and low-throughput due to their reliance on pumps for medium circulation. We have developed a simple multi-organ MPS device, namely interconnected multi-well device, that utilizes a three-dimensional shaker for medium circulation, eliminating the need for pumps. The device consists of two components: a set of four connected cell-culture cups and a series of four interconnected wells. Up to six devices can fit in a standard 24-well plate. The device supports various culture methodologies, including conventional two-dimensional culture and spheroid culture, and is able to accommodate cell culture inserts. Here, we demonstrated the circulation of medium and immune cells throughout the device. Then, as a representative use-case scenario, we demonstrated using the device to evaluate the anticancer effects of the prodrug capecitabine, whose metabolite exhibits anticancer effects, in a two-organ system composed of liver and cancer. In short, our interconnected multi-well device is user-friendly, adaptable to various culture methods, and multi-throughput, and it shows promise for becoming a valuable tool for in vitro organ interaction research.

Recently, sake aged for several years to decades, called long-term aged sake, has attracted some attention. Aged liquors also exist in other countries, such as Chinese rice wine (Huangjiu) and fortified wines (Sherry, Madeira, Port). To understand the characteristics of long-term aged sake among the typical aged liquors worldwide, we analyzed and compared the flavor components and sensory characteristics of aged sake, Huangjiu, and fortified wines. Accordingly, flavor component analysis revealed a common trend toward increases in sotolon, aldehydes, polysulfides, ethyl esters, and decreases in acetate esters with age. However, the increases in aldehydes and polysulfides were more significant in sake and Huangjiu, whereas the increases in ethyl esters were more significant in fortified wines. Moreover, the concentrations and variances of acetate esters in sake were highest among the five liquor categories. Sensory evaluation revealed that sake and Huangjiu presented stronger sulfur aroma and bitter taste, whereas fortified wines presented stronger dried fruit, honey aroma and sweet taste. Furthermore, sake samples showed a larger variance in the intensity of caramel, burnt, and soy sauce aroma than did the other liquor categories. These results indicate that the compositional and sensory characteristics of the aged liquors can be grouped into sake/Huangjiu and fortified wines and that sake is featured by extensive variations in the levels of age-related components and sensory attributes.

Chicken primordial germ cells (cPGCs) hold great potential for genetic modification and germ cell research in chickens. In this study, we evaluated the cellular characteristics of three cPGC lines: cPGC-1, cPGC-2, and cPGC-3. cPGC-1 and cPGC-2 were derived from male chickens, whereas cPGC-3 was derived from a female chicken. We analyzed and compared cell proliferation rates, marker gene expression, and gonadal colonization abilities. Three different cell culture temperatures were assessed (37 °C, 39 °C, and 41 °C) and proliferation rates were highest for all cPGC lines at 39 °C. Additionally, cPGC-1 demonstrated a higher proliferation rate than cPGC-2. No significant differences were observed between cPGC-1 and cPGC-2 with regard to the expression of germ cell and pluripotency marker genes (Cvh, Dazl, Pou5f3, and Nanog). To assess changes in cellular characteristics before and after genetic modification, we performed a green fluorescent protein (GFP) gene knock-in using the CRISPR/Cas9 system, followed by site-specific integration of the scFv-Fc gene using the Cre-loxP system. Transplantation experiments revealed that cPGC-2/GFP exhibited higher gonadal colonization efficiency than cPGC-1/GFP. This study demonstrates differences in cellular characteristics among established cPGC lines and highlights the impact of genetic modification on cPGC function. Our findings emphasize the importance of selecting appropriate cell lines and optimizing culture conditions based on cPGC traits to achieve efficient and reproducible production of transgenic chickens. These insights will aid in the conservation of poultry genetic resources and the advancement of transgenic chicken production for both research and industrial applications.

Polyhydroxyalkanoates (PHAs) are polyesters accumulated by various microorganisms as intracellular inclusions. In this study, Escherichia coli was engineered to co-express PHA biosynthetic genes and the ratiometric fluorescent biosensor QUEEN. QUEEN is a genetically encoded fusion protein comprising green fluorescent protein and the ε subunit of ATP synthase, which functions as an ATP-binding domain. Its fluorescence intensity ratio at two wavelengths reflects intracellular ATP levels. In this study, we aimed to monitor in situ ATP levels during poly(3-hydroxybutyrate) [P(3HB)] production in E. coli. The metabolic pathway was designed to initiate and enhance P(3HB) synthesis through precursor (3HB) supplementation, while PHA biosynthetic genes were constitutively expressed. Consequently, P(3HB) production was successfully induced in recombinant cells harboring the QUEEN gene, with 78 % of Nile Blue-stained cells exhibiting PHA inclusions under microscopy. QUEEN remained functionally expressed under both P(3HB)-producing and non-producing conditions. Single-cell fluorescence measurements using QUEEN revealed no significant difference in ATP levels between the two conditions. These results suggested that E. coli possesses homeostatic functions in energy metabolism during PHA production.

Acetic acid bacteria convert environmental sugars and alcohols into acetic acid and various sugars through oxidative fermentation, resulting in the accumulation of these compounds at high concentrations in the culture medium. One such product is the rare sugar 5-keto-d-fructose (5-KF). In Gluconobacter species, 5-KF is transported into the cell and reduced to fructose in a single step by 5-ketofructose reductase, allowing entry into glycolysis. However, it remains unclear whether eukaryotic microorganisms can metabolize 5-KF or which genes are involved in this process. In this study, we investigated the ability of various yeasts to utilize 5-KF and identified genes involved in its metabolism. The model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were unable to grow on 5-KF, whereas the oleaginous yeast Lipomyces starkeyi efficiently metabolized this sugar. RNA-seq analysis of L. starkeyi grown on 5-KF revealed genes specifically upregulated in response to 5-KF. Based on gene annotation and expression profiles, a putative metabolic pathway was proposed. Gene knockout analyses showed that mutants deficient in specific steps of the pathway grew on downstream intermediates but failed to grow on upstream substrates, indicating loss of the corresponding enzymatic functions. These results suggest that L. starkeyi metabolizes 5-KF via a multistep pathway, 5-KF → l-sorbose → d-sorbitol → d-fructose. This study provides the first evidence of a 5-KF metabolic pathway in yeast, distinct from the single-step conversion to fructose observed in Gluconobacter species.

Pancreatic cancer remains one of the most lethal malignancies, with a 5-year survival rate of less than 9 %, primarily due to its aggressive metastasis and drug resistance. Circulating tumor cells (CTCs), which are key mediators of metastasis, are critical for understanding these clinical challenges. In this study, we applied a single-cell transcriptome analysis platform combining a microcavity array (MCA) and gel-based cell manipulation (GCM) technique for marker-independent cell recovery to analyze CTCs from patients with metastatic pancreatic cancer. Using pancreatic cancer cell lines, we demonstrated that this microcavity-gel manipulation platform enables high-quality single-cell RNA sequencing while preserving intrinsic molecular characteristics. Furthermore, spiking experiments with cancer cells in blood confirmed that the process minimizes contamination from non-target blood cells. Application of this platform to patient-derived CTCs revealed that most CTCs exhibited epithelial-mesenchymal transition (EMT)-like features and high expression of platelet-related genes such as PF4 and PPBP, suggesting platelet-driven EMT activation. In addition, CTCs were consistently arrested in the G1 phase of the cell cycle, implying a potential survival mechanism under therapeutic stress. These findings highlight the utility of microcavity-gel manipulation platform for robust single-cell transcriptomic profiling and provided novel insights into the biology of pancreatic CTCs.