Journal of bioscience and bioengineering最新文献

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.

Sago is a high-carbohydrate, naturally gluten-free product derived from tropical palm trees, serving as an essential staple food in many regions of Southeast Asia and the Pacific Islands. The various processing methods used to produce sago starch can influence its overall quality, including its flavor. To date, a comprehensive analysis of its flavor quality across different processing methods has not yet been conducted. This study aimed to characterize the flavor quality of sago starch by combining physicochemical, metabolomic, and sensory analyses. Sago starch samples produced using traditional, semi-mechanized, and modern methods were collected and analyzed. Principal component and heatmap analyses revealed that traditional processing resulted in lower sensory and physicochemical quality, characterized by higher off-flavor compounds, particularly organic acids, likely due to uncontrolled microbial activity. In contrast, modern processing yielded higher levels of sugars such as sucrose and fructose, associated with desirable flavor, while semi-mechanized processing produced intermediate flavor profiles, possibly due to partial fermentation. Partial least squares regression analysis identified potential key metabolites related to flavor deterioration in sago starch, including octanoic acid, 3-methylbutyric acid, and hexanal. These findings can support improvements in sago starch processing to enhance flavor quality and guide quality control strategies in the industry.

This study presents a preliminary report on the development of rabbit monoclonal antibodies targeting the extracellular domain of rat G protein-coupled receptor 54 (GPR54). The extracellular domain of GPR54 (GPR54 ECD) was fused with the albumin-binding domain of Streptococcus sp. protein G, and subsequently expressed in Escherichia coli, purified, and used to immunize a rabbit. Antigen-specific B cells were isolated using fluorescently labeled peptides corresponding to the GPR54 extracellular domain 2 region. Antibody genes were amplified from the sorted B cells, cloned into vectors, and transformed into competent cells. Combinatorial pairing of light and heavy chain genes, followed by cell-free protein synthesis, led to the identification of four antibody pairs with reactivity toward the GPR54 ECD based on enzyme-linked immunosorbent assay. Western blotting confirmed the ability to detect target proteins with minimal cross-reactivity. This study highlights the possible use of the combinatorial pairing of antibody genes to isolate rare antigen-specific monoclonal antibodies.

Poly(ε-caprolactone) (PCL), also known as poly(6-hydroxyhexanoate) [P(6HHx)], is a biodegradable polyester characterized by excellent flexibility, processability, and marine degradability, making it a promising alternative to conventional plastics. However, current chemical syntheses of PCL rely on metal-catalyzed ring-opening polymerization of ε-caprolactone, raising concerns about metal contamination and environmental sustainability. Here, we report a biological method to synthesize PCL [P(6HHx)] using an engineered polyhydroxyalkanoate (PHA) system in Escherichia coli. An artificial PHA synthase (PhaC), FcPhaC4, designed via a full-consensus design algorithm to enhance structural stability and broaden substrate specificity, was employed for polymer production. E. coli expressing FcPhaC4 and cultivated with the supplementation of 6HHx synthesized polymer, confirmed to be PCL by ¹H/¹³C Nuclear Magnetic Resonance and Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry analyses. The F313Y mutant of FcPhaC4 further improved PCL yield. In addition, using these enzymes, random copolymer P(3-hydroxybutyrate-co-6HHx) was synthesized at desired monomer compositions. In vitro assays demonstrated that FcPhaC4 and its mutant exhibited activity toward 6HHx-CoA as a sole substrate, being consistent with their homopolymer-producing capacity. These results indicated that FcPhaC4 is the first enzyme capable of biologically synthesizing PCL homopolymer.

Three-dimensional engineered muscle (3D-EM) provides a physiologically relevant model for examining skeletal muscle function. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine elevated in chronic conditions such as sarcopenia and cachexia, has been linked to muscle weakness. However, the mechanism underlying this effect remains unclear. In this study, we used a 3D-EM system to evaluate the direct impact of TNF-α on muscle contractile force. 3D-EM was produced by seeding C2C12 myoblasts with type I collagen on a culture device, followed by 15 days of differentiation. Constructs were then treated with TNF-α for 48 or 72 h, and contractile output was measured during electrical pulse stimulation. Immunohistochemical analysis and RNA sequencing (RNA-seq) with subsequent enrichment analysis were conducted to assess tissue structure and transcriptomic changes. After 48 h, TNF-α reduced contractile force by 60 %, and after 72 h, by 90 % relative to controls. Immunohistochemistry showed myotube atrophy accompanied by loss of fast-twitch fibers. RNA-seq combined with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated suppression of extracellular matrix, sarcomere organization, and calcium signaling pathways. These results suggest that TNF-α reduced force generation in 3D-EM by impairing extracellular matrix integrity, sarcomeric structure, and calcium-dependent contraction mechanisms, with preferential effects on fast-twitch fibers. Overall, this study offers mechanistic insight into the basis of sarcopenia and demonstrates the utility of 3D-EM as a model of cytokine-induced muscle weakness.

The yeast Kluyveromyces marxianus assimilates various sugars, including sorbitol and mannitol. However, the metabolic pathways for sugar utilization, including sugar transporters, remain to be elucidated. To identify these genes in this study, first 13 candidate transporter genes were disrupted using a newly developed non-homologous end joining (NHEJ)-mediated gene disruption method, combined with targeted digestion using the CRISPR-Cas9 system. While most disruptants exhibited no clear growth defects in various sugar media, a disruptant of the KmMLEV2025 gene (named KmSAT1) failed to grow in either sorbitol or mannitol media, suggesting that it encodes a sugar alcohol transporter. Next, we investigated the candidate dehydrogenase genes crucial for sugar alcohol metabolism, as they are converted to fructose by dehydrogenases. KmXyl2p, a known xylitol dehydrogenase, is a candidate sorbitol dehydrogenase. Disruption of KmXYL2 caused growth defects in sorbitol medium, but not in mannitol medium. We disrupted several genes to identify the mannitol dehydrogenase, revealing that the disruption of KmSOU2, annotated as a sorbose reductase, resulted in a growth defect in the mannitol medium. The identified genes were overexpressed for the efficient utilization of sugar alcohols. The strain overexpressing KmSAT1, but not the dehydrogenase genes, started growing immediately, whereas the wild-type strain exhibited a lag time of several days. Furthermore, the final cell optical densities in both the sorbitol and mannitol media were higher than those observed in the glucose medium. These results indicated that overexpression of a sugar alcohol transporter is a highly effective strategy for biotechnological applications.