Biotechnology and Bioprocess Engineering最新文献

Cellulose is widely considered an outstanding biomaterial due to its remarkable ionic properties, exceptional biocompatibility, and low toxicity. Its abundant surface hydroxyl groups facilitate increased hydrogen bonding, improving gelation and swelling capabilities. Moreover, incorporating carboxymethyl groups enhances solubility and allows for diverse formulations, serving as multifunctional cross-linkers. Among the various sources of this compound, tunicate-derived cellulose is an animal-derived cellulose and food byproduct with low utility. However, recycling tunicate skin into a useful biomaterial would provide access to the unique characteristics of animal cellulose, distinct from those of plant-derived cellulose. Particularly, tunicate cellulose has a longer fiber length than plant cellulose, enhancing the sound propagation speed within the material and making it suitable for the production of ultrasound-responsive gels. This study examined the viscosity and conductivity of tunicate-derived carboxymethyl celluloseto assess its applicability as an ultrasound gel. Additionally, small molecule release after ultrasound stimulation was also evaluated.

Organic–inorganic biohybrids have recently garnered attention for biomedical applications owing to their outstanding catalytic activity and biocompatibility. However, their efficacy in enhancing specificity toward drug targets remains limited. Here, we developed a transferrin–doxorubicin (TRF–DOX) complex and green fluorescence protein (GFP)-conjugated copper (Cu) phosphate (TRF–DOX@GFP@Cu biohybrid) for use as an imageable drug delivery system (DDS). TRF was utilized to increase the affinity of drug carriers for TRF receptors on cancer cells, and DOX was selected as a model drug. Additionally, GFP provides fluorescence properties for bioimaging and Cu ions serve as the skeleton for forming the flower-shaped inorganic material. By adjusting the concentrations of TRF–DOX and GFP with 25 mg mL⁻¹ of Cu precursors, six flower-shaped TRF–DOX@GFP@Cu biohybrids were fabricated. Among these, biohybrid-5 (prepared using 0.05 mg mL⁻¹ TRF–DOX and 0.10 mg mL⁻¹ of GFP with 25 mg mL⁻¹ of Cu ions) exhibited the strongest fluorescence. We characterized the morphology, composition, functional groups, and specific surface area of the TRF–DOX@GFP@Cu biohybrid. Biohybrid-5 had a specific surface area of 37.508 m² g⁻¹ and could effectively bind to A549 lung cancer cells as shown by fluorescence imaging. The novel TRF–DOX@GFP@Cu biohybrid fabricated in this study has potential as a DDS in the treatment of lung cancer.

A robust cell recycling strategy based on the methylotrophic yeast, Pichia pastoris, was established to enhance product titers of the commercially important Yarrowia lipolytica lipase Lip2. The expression of Lip2 protein in the prokaryotic host Escherichia coli resulted in inclusion bodies, whereas utilization of SUMO fusion tag could not improve its solubility. Therefore, Lip2 extracellular expression was targeted via the Saccharomyces cerevisiae α-mating signal sequence in P. pastoris under methanol-inducible AOX1 promoter. Shake flask expression studies of hyper-producer Pichia clone under optimized conditions resulted in 438.83 and 420.09 mg/L of glycosylated Lip2 production after induction at an OD₆₀₀ of 10 and 20, respectively. A high Lip2 productivity was further targeted using a cell retention technique where the cell biomass was recycled to obtain higher product concentration with improved product quality. The biomass recycling at every 72 h followed a 3.8-fold enhanced Lip2 concentration with a cumulative volumetric product concentration of 1,794 mg/L. A high specific product yield (Y_P/X) in the range of 37.45–47.00 mg/g dry cell weight (DCW) was also maintained up to five retention cycles. Furthermore, higher cumulative protein yields were obtained from the 5-time recycled cells compared to five individual batch runs at shake flask up to 72 h. High cell density cultivation of recombinant P. pastoris in a 2.5-L fermenter yielded 5.25 g/L of Lip2 enzyme with a maximum specific yield of 51.97 mg/g DCW.

The presence of antibodies (Abs) in the body is an indicator of the individual immune response to an infection. In the post-pandemic era, an accurate, cheap, and rapid antibiotic sensor is critically needed to monitor Ab levels. Therefore, we present a simple fabrication method for a highly sensitive electrochemical sensor that detects SARS-CoV2 spike Abs. The sensor comprises filter paper with a composite made of polypyrrole (PPy) and reduced graphene oxide (rGO). The rGO structure is observed using a scanning electron microscope and Fourier-transform infrared spectra (FTIR). The presence of rGO improves protein conjugation which is identified using a fluorescent microscope and FTIR. The sensitivity test performs high selectivity and reproducibility in two different SARS-CoV2 Abs separately and shows the range of detection limits between 10 and 1000 fg/mL. The results demonstrate an advancement in developing PPy/rGO composite sensors for SARS-CoV2 Ab detection and as a promising sensor for monitoring COVID-19 exposures and infections.

Chinese hamster ovary (CHO) cells play an important role in the biopharmaceutical industry, but their production efficiency requires enhancement to meet the growing market demands. Artificial intelligence (AI) has been a potent tool for modeling bioprocesses to support biopharmaceutical manufacturing. However, existing AI models do not adapt well to process data collected at irregular time intervals and have limited capability to scale up and down to incorporate various process parameters. To address the limitations, this study develops a novel neural ordinary differential equation (ODE) model for predicting key variables such as viable cell concentration, glucose concentration, lactate concentration, pH, and dissolved oxygen in a CHO cell bioreactor. Validated through extensive bioreactor experiments, the neural ODE model shows a better accuracy compared to the benchmark models, which include a conventional mechanistic model and a hybrid model. Additionally, the neural ODE model incorporated essential process variables that were not considered in the previous models. It successfully extrapolates to predict unknown dynamics at different initial conditions, which showcases robust adaptability. Moreover, the model provides useful insights into the relationship among variables, highlighting its potential for bioprocess modeling.

Polyhydroxyalkanoates (PHAs), one of biodegradable polyesters, are substances that store carbon and energy in various microorganisms. Polyhydroxybutyrate (PHB) is the most commonly type of PHAs with relatively simple chemical structure. In this study, we developed a polymerase chain reaction (PCR) screening method for screen novel halophilic bacteria containing PHA synthase (PhaC) gene. Halophilic bacteria were collected and isolated from high-salt environment such as sea soil or shrimp jeotgal in South Korea. Primer set was designed based on the conserved region in Class I PhaC from 30 kinds of Halomonas species. The designed primer set was used to optimize PCR conditions to identify PhaC gene in newly isolated 15 halophilic bacteria from sea soil or shrimp jeotgal. Among 15 candidates, five bacteria were selected after PCR and agarose gel analysis and confirmed to produce PHB. Two bacteria with higher PHB production were identified as Halomonas and Marinobacter sp. by 16 s rRNA analysis and named as Halomonas shrimpha IBTH01 and Marinobacter haeunpha IBTM02. Then, PHB production was examined by changing culture temperature, media composition, carbon source, and glucose concentration. Finally, PHB from two bacteria was analyzed by transmission electron microscopy, gas chromatography, and ¹H nuclear magnetic resonance. Taken together, this study will contribute to establish a platform for the utilization of novel halophilic bacteria in the synthesis of PHA polymers.

Fermentation products and lysates of lactic acid bacteria (LAB) have been developed as cosmetic ingredients. The topical application of certain LAB strains can improve skin health and combat skin diseases. Here, we investigated the effects of nano-sized lysate of Lactiplantibacillus plantarum APsulloc 331261 (NLAP) isolated from green tea leaves on human skin cells and a reconstructed human epidermis (RHE) model. NLAP increased the expression of genes involved in skin barrier functions such as proliferation, differentiation, tight junction formation, and antimicrobial peptides in normal human epidermal keratinocytes. NLAP prevented the decrease in the expression of differentiation markers and increased release of inflammatory cytokines in keratinocytes cultured with Staphylococcus aureus. NLAP-induced improvements in gene expression and cytokine levels were also observed in RHE treated with heat-killed S. aureus. Additionally, the skin barrier-strengthening effect of NLAP was confirmed by comparing the penetration of the fluorescent dye into the RHE. These findings suggest that NLAP could aid skin barrier function, protect the skin against detrimental bacteria, and suppress inflammatory responses; thus, it can be developed as a skincare ingredient.

In this research, we investigated a naturally occurring, non-genetically modified strain of Acinetobacter sp., isolated from soil, which demonstrated the capability to produce both indigo and biosurfactant. During the screening, indole was used as the sole carbon source in M9 minimal medium. The strain exhibiting the most intense blue coloration was isolated and further analyzed. The blue dye extracted from the cell culture was confirmed as indigo through LC/MS analysis, showing an m/z value of 263.5, and H-NMR analysis. In LB medium, the wild-type Acinetobacter sp. strain produced approximately 6.8 mg/L of indigo from 1 mM indole. However, in M9 minimal medium, the production yield significantly increased to 45.5 mg/L. Notably, the isolated strain showed vigorous bubbling during growth, which could facilitate the transport of indole and indigo dye, both of which have low solubility, across cell membranes. Additionally, this strain was capable of degrading medium-chain C12 alkane efficiently. The whole genome was fully sequenced and analyzed for genes concerning biosurfactant and alkane metabolisms. In conclusion, utilizing a wild-type strain for indigo production offers a promising alternative to traditional chemical processes, addressing concerns related to genetically modified organisms in future applications.

Few studies have investigated the biodegradation of microplastics in marine environments. Microorganisms that can degrade microplastics in high-salinity conditions are sought after. Therefore, we aimed to isolate a halotolerant poly(ε-caprolactone) (PCL)-degrading bacterium for applications in biotechnology. The bacterium isolated from seaside soil was identified as Bacillus gibsonii via phylogenetic analysis based on 16S rRNA gene sequences and designated as KRICT-1. We tested whether the KRICT-1 strain showed halotolerance by determining the sodium chloride (NaCl) tolerance at various concentrations. The KRICT-1 strain showed growth at up to 10% NaCl on Luria–Bertani (LB) medium agar plates and 10% NaCl in liquid LB medium, indicating that KRICT-1 can grow and produce a PCL-depolymerizing enzyme under high-salt conditions. The KRICT-1 strain could depolymerize PCL with a PCL film weight loss of 2.82% at up to 10% NaCl concentration after cultivation of 7 weeks. KRICT-1 is the first strain of B. gibsonii which shows PCL-depolymerizing activity. Scanning electron microscopy and water contact angle results confirmed the degradation of PCL by the KRICT-1 strain. The extracellular enzyme produced by the KRICT-1 strain was stable over a wide range of temperatures (15–40 °C) and pH (7.0–9.5). This halotolerant PCL-degrading bacterium can be used in the degradation of biodegradable plastics present in saline soils, saline water, and wastewater.

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Patients with triple-negative breast cancer (TNBC) often face an unfavorable prognosis due to the lack of targeted therapy. Thus, identifying drug targets specific to the TNBC subtype is crucial for effective treatment. Here, we propose a strategy to identify potential inhibitory targets specific to this subtype based on the gene dependency score (GDS), a quantitative measure of essentiality of each gene determined in cancer cell lines. Specifically, we compared the GDS values of 17,387 genes among cell lines of four breast cancer (BC) subtypes, namely luminal A, luminal B, HER2-positive, and TNBC, to identify genes showing specific essentiality in TNBC subtype cell lines. Twenty-two genes were predicted as potential inhibitory targets. Of these, we selected two genes, ILK and RHOA, based on survival analysis conducted across the four BC subtypes. We propose these two genes as potential biomarkers for TNBC. Furthermore, we experimentally validated that inhibiting ILK expression with a specific inhibitor reduced cell viability more in TNBC subtype cell lines than in other BC subtype cell lines. Therefore, ILK is a potential drug target specific to TNBC. The strategy proposed is expected to be useful in identifying biomarker and therapeutic target genes in not only BC but also other cancers.