Journal of bioscience and bioengineering最新文献

In soy sauce brewing, the halophilic lactic acid bacterium, Tetragenococcus halophilus is used as a fermentation starter and contributes to the taste and aroma of soy sauce, mainly by producing lactate. By lowering the pH of the soy sauce mash, lactate serves as a suitable growth environment for the halotolerant yeast Zygosaccharomyces rouxii. Acetate, which is produced by T. halophilus via the citrate metabolic pathway, is a critical growth inhibitory factor for Z. rouxii. Therefore, a T. halophilus strain that lacks acetate production could be an ideal fermentation starter to enhance ethanol production. In this study, we obtained a derivative of T. halophilus containing an insertion sequence in citC, which is an essential gene for citrate metabolism, and validated its performance as a soy sauce fermentation starter. The derivative neither metabolized citrate nor produced excessive acetate in soy sauce mash, resulting in vigorous alcohol fermentation by Z. rouxii. This study provides insights into the application of a low acetate-producing strain of T. halophilus as a starter to produce soy sauce with high alcohol content and low sour aroma.

Non-invasive cell culture monitoring technology is crucial to improve the manufacturing efficiency of cell products. We have found that extracellular vesicles (EVs) are secreted into the culture supernatants in the differentiation process from human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells, and that the composition of EVs changes in accordance with the differentiation processes. In this study, we hypothesized that it is possible to evaluate the cultured cellular states by detecting compositional changes of EVs secreted from cultured cells with label-free Raman spectroscopy in a non-invasive manner. Therefore, Raman signal analysis derived from EV fractions isolated from culture supernatants throughout the differentiation process was conducted. iPSCs cultures were simultaneously implemented under a standard condition (control) and an artificial deviation condition inducing reductions in pluripotency by depleting FGF2 in culture medium (-FGF2), which is indispensable for maintaining the pluripotency. Subsequently, the differentiation step was conducted for each iPSCs culture under the same condition. As a result, it was found that under -FGF2, the expression level of the pluripotency marker NANOG decreased compared to that of the control and correlated with the identification results based on Raman signals with a correlation coefficient of 0.77. Lipid-derived Raman signals were extracted as identification factors, suggesting that changes in the lipid component of EV occur depending on the cellular states. From the above, we have found that the change in composition of EVs in the culture supernatant by detecting Raman signals would be a monitoring index of the cellular state of differentiation and pluripotency.

Resolvin E series (Rvs), such as RvE4 (5S,15S-dihydroxyeicosapentaenoic acid) and its stereoselective enantiomer (5R,15R-dihydroxyeicosapentaenoic acid), play an important role in promoting the resolution of inflammation and are derived from eicosapentaenoic acid (EPA) by M2 macrophage in human. However, they have been synthesized using expensive and inefficient chemical methods. Here, we performed efficient quantitative production of RvE4 and its enantiomer from EPA using Escherichia coli expressing double-dioxygenating 15S-lipoxygenase (15S-LOX) from Archangium violaceum and double-dioxygenating 15R-LOX from Sorangium cellulosum, respectively, with solvent, polymer, and adsorbent resin. The cell density, substrate concentration, solvent types and concentrations, polymer types and concentrations, and resin concentration were optimized for the enhanced bioconversion of EPA into RvE4 and its enantiomer. Under the optimized conditions, A. violaceum 15S-LOX and S. cellulosum 15R-LOX expressed in E. coli converted 6.0 mM EPA into 4.3 mM (1.44 g/L) RvE4 and 5.8 mM (1.94 g/L) RvE4 enantiomer in 60 min, with productivities of 4.3 and 5.8 mM/h and molar conversions of 72% and 97%, respectively. To date, these are the highest concentrations, productivities, and conversions of RvE4 and its enantiomer. The concentrations of RvE4 and its enantiomer obtained from the conversion of EPA with solvent, polymer, and resin were 2.5- and 3.2-fold higher than those without the additives, respectively.

Aiming to build a tissue analogue engineered cartilage from differentiated chondrocytes, we investigated the potential of a pyrocarbon (PyC)-based and scaffold-free process, under mechanical stimulation. PyC biomaterial has shown promise in arthroplasty and implant strategies, and mechanical stimulation is recognized as an improvement in regeneration strategies. The objective was to maintain the cell phenotype to produce constructs with cartilage-like matrix composition and mechanical properties. Primary murine chondrocytes were deposited in drop form between two biomaterial surfaces expanded to 500 μm and a uniaxial cyclic compression was applied thanks to a handmade tribo-bioreactor (0.5 Hz, 100 μm of amplitude, 17 days). Histology and immunohistochemistry analysis showed that PyC biomaterial promoted expression of cartilage-like matrix components (glycosaminoglycans, type II collagen, aggrecan). Importantly, constructs obtained in dynamic conditions were denser and showed a cohesive and compact shape. The most promising condition was the combined use of PyC and dynamic stimulation, resulting in constructs of low elasticity and high viscosity, thus with an increased damping factor. We verified that no calcium deposits were detectable and that type X collagen was not expressed, suggesting that the cells had not undergone hypertrophic maturation. While most studies focus on the comparison of different biomaterials or on the effect of different mechanical stimuli separately, we demonstrated the value of combining the two approaches to get as close as possible to the biological and mechanical qualities of natural hyaline articular cartilage.