Laccases have shown to be efficient biocatalysts for the removal of recalcitrant pollutants from wastewater. Thus, they catalyse the oxidation of a wide variety of organic compounds by reducing molecular oxygen to water. However, the use of free laccases holds several drawbacks such as poor reusability, high cost, low stability and sensibility to different denaturing agents that may occur in wastewater. Such drawbacks can be circumvented by immobilising laccase enzymes in/on solid carriers. Hence, during the last decades different approaches considering various techniques and solid carriers to immobilise laccase enzymes have been developed and tested for the removal of pollutants from wastewater. To scale up wastewater treatment bioprocesses, the immobilised laccases are placed in different reactor configurations.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed E. coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg/OD600. As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. The AAV purification pipeline, in particular, hinges on protein ligands for the affinity-based capture step: while featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product’s activity and stability; additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable – yet equally effective – affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (~1014 vp per mL) and product yields (~60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified vectors.

Statins as a lipid-lowering drug can selectively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and decrease cholesterol synthesis effectively. With the improvement of nutritional conditions, the demand for statins is increasing in global market. Due to the rapid development of modern biotechnologies, the biosynthesis of stains by microbial cell factory appears great advantages. It has the advantages of simple operation and easy separation of products. This review summarized the strategies on statins production via microbial cell factory, including both traditional fermentation culture and modern synthetic biology manufacture. Firstly, the complex fermentation parameters and process control technology on submerged fermentation (SmF) and solid-state fermentation (SSF) were introduced in detail. Especially, the possibility of recoverable agricultural wastes/(Biomass) as fermentation substrate on solid-state fermentation to produce statins was emphasized. Besides, metabolic engineering strategies to construct robust engineering strains and strains evolution were also discussed. The review highlights the potential and challenge of microbial cell factory to yield the statins. Thus, it will facilitate the production of statins in more green production mode.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction. This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies/µl; that of genomic samples was 0.1 ng/µl. The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1 h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n=93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing.

Background Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. Methods and major results Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome Mosaic Virus (BMV) and containing the catalytic activity of glucose oxidase enzyme (GOx). The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactors treatement on triple negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. Conclusions and implications The nanoreactors containing GOx activity are fully suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors could result in a prevailing strategy to improve selective cancer targeting. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are on going to reinforce the effectiveness of this treatment strategy.

Liposomal drug products are playing an increasing role in the field of drug delivery. With this increased demand comes the need to increase the capabilities and capacity of manufacturing options. Continuous manufacturing techniques present a significant opportunity to address these needs for liposomal manufacturing processes. Liposomal formulations have unique considerations that impact translation from batch to continuous process designs. This article examines aspects of converting to a continuous design that were previously viewed as inconsequential in a batch process. The batch process involves the removal of ethanol through tangential flow filtration (TFF). Ethanol was found to reduce the permeability of the hollow fibers used for TFF. This effect was determined to have minimal impact on the overall batch process design but considerable influence on the design of continuous TFF such as inline diafiltration (ILDF). Using a pilot scale setup, ethanol was found to decrease permeability in an inverse manner to ethanol concentration. Further assessment found that dilution of the ethanol levels prior to diafiltration can significantly reduce the amount of ILDF stages needed and that a continuous design requires less buffer to the commensurate batch design.

Microtiter plates are suitable for screening and process development of most microorganisms. They are currently the container of choice for high-throughput and small-scale microbial culture, but require optimization for specific work. This research presents a novel type of microtiter plate was developed using computational fluid dynamics (CFD) technology. The new plate provides high oxygen supply and optimal mixing effects for the fermentation culture of docosahexaenoic acid (DHA) producing strains, surpassing the conventional method of strains screening with shake flasks, which is insufficient. the shape of the microtiter plate was modified, and baffles were introduced to improve mass transfer and oxygen supply effects in the vibrating bioreactor. CFD technology was used to model the new plate’s characteristics, establishing the superiority of hexagonal microtiter plates with six baffles. Parameters in the incubation process, such as vibration frequency and liquid load, were optimized, and the final result achieved a KLa of 0.61s-1 and a volume power input of 2364 w/m3, which was 4-5 times better than the original 96-well plate. The culture results optimized by the model were also verified. Therefore, this new microtiter plate provides a powerful tool for future high-throughput screening of strains.

In the last two decades, there have been significant advancements in the development of more physiologically relevant organ-on-a-chip (OOC) systems that can mimic the tissue microenvironment. Despite the advantages of these microphysiological systems, such as portability, the ability to mimic physiological flow conditions, and the reduction of reagents required for preparation and detection, they lack real-time detection of analytes with high accuracy. To address this, biosensor technologies have been integrated with OOC systems to enable simultaneous analysis of different analytes in a single device. However, integrating biosensors with OOC systems is challenging due to the competing demands for low-cost, simple fabrication processes, and speed. This study presents the fabrication of a glucose sensing device integrated with a liver-on-a-chip (LOC) platform. The conductive PLA-based three-electrode system was printed using FDM 3D printing technology to simplify the fabrication process. The sensitivity of the glucose biosensing device was enhanced by adding multi-walled carbon nanotubes on the electrodes. The biosensing integration study using a perfusion-based LOC showed the stability, biocompatibility, and sensitivity of the glucose sensing devices. Furthermore, drug toxicity studies on the LOC platform demonstrated the device’s ability to detect a broad range of glucose concentrations and its enhanced sensitivity.

The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted in vivo mutagenesis system to significantly increase mutation frequency and generate mutations across all four nucleotides. We constructed different DNA-modifying enzyme-PmCDA1-T7 RNA polymerase fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-modifying enzymes. With the mutagenesis fusion proteins, the mutation frequency of the target gene could reach 5.13x10-3, and the proportion of non-C→T mutations is 10~11-fold higher than the cytidine-based evolutionary tools. We also demonstrated that our mutagenesis tools could be used to evolve the essential enzyme in the β-carotene biosynthesis process and generate mutations with different types.

Designing and selecting cell culture media and their feeding are a key strategy to maximize culture performance in industrial biopharmaceutical processes. However, mammalian cells are very sensitive to their culture environment, requiring specific nutritional needs to grow and produce high-quality proteins such as antibodies, depending on cell lines and operational conditions. In this regard, previously we developed data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research further, we evaluated two chemically defined feed media, A and B, in ambr15 bioreactor runs using a monoclonal antibody-producing CHO K1 cell line. We observed a significant impact of feed media on cell growth, longevity, viability, productivity and toxic metabolites production. Specifically, concentrated feed A was not sufficient to support prolonged cell culture and high titer compared to feed B. The framework systematically characterized the major metabolic bottlenecks in the TCA cycle and its related amino acid transferase reactions, thereby identifying key design components, such as asparagine, aspartate, and glutamate, which are needed for highly productive cell cultures. Based on our results, we subsequently reformulated the feeds by adjusting the amounts of those amino acids and successfully validated their effectiveness in promoting cell growth and/or titer.

Development of disposable, rapid and convenient biosensor with high sensitivity and reliability is the most desired method of viral disease prevention. To achieve this goal, in this work, a practical impedimetric biosensor has been implemented into a disposable electrode on a screen-printed carbon electrode (SPCE) for the detection of two mosquito-borne viruses. The biosensor fabrication has step-wisely carried out on the disposable electrode surface at room temperature: starting from conductive film formation, physical binding of the gold nanoparticles (AuNPs)-polyaniline (PAni) into the conductive film, and biofunctionalization. To get the maximum efficiency of the antibody, biotinylated antibody has been conjugated on the surface of AuNP-PAni /PAni-SPCE via the streptavidin-biotin conjugation method which is a critical factor for the high sensitivity. Using the antibody-antigen interaction, this disposable electrode has designed to detect mosquito-borne infectious viruses, Chikungunya virus (CHIKV) and Zika Virus (ZIKV) separately in a wide linear range of 100 fg/mL to 1 ng/mL with a low detection limit of 1.33 fg/mL and 12.31 fg/mL, respectively.

α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) from L-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. Main Methods and Major Results This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild type, Isothermal Titration Calorimetry(ITC) results showed that the equilibrium dissociation constant (KD) of the mutant for AA-2G was doubled; the Michaelis constant (Km) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. Conclusions and Implications Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. So, improving the tolerance of enzymes in organic solvents is essential. Herein, the method of regional random mutation combined with combinatorial mutation was used to improve the resistance of transaminase from Aspergillus terreus (AtATA) in organic solvents, and the best mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency toward 1-acetylnaphthalene and the stability were higher than the wild-type (WT) of AtATA. M3 also showed enhanced stability against six organic solvents with different oil-water partition coefficients (log P values). The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics simulations under different concentration DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Intra- and intermolecular interaction analysis indicated that the increased hydrogen bonds and hydrophobic interactions within monomers or at the interface of two monomers also strengthened the stability of the overall structure against organic solvents. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was and an intermediate of Cinacalcet Hydrochloride. Moreover, 3~10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 94.2~38.9% yield and a strict R-stereoselectivity within 10 h under 25% DMSO, which was higher than WT and expected to be a potential biocatalyst for industrial application.

Periosteum has shown potential as an effective barrier membrane for guided bone regeneration (GBR). However, if recognized as a “foreign body”, insertion of a barrier membrane in GBR treatment will inevitably alter the local immune microenvironment and subsequently influence bone regeneration. The aim of this investigation was to fabricate decellularized periosteum (DP) and investigate its immunomodulatory properties in GBR. DP was successfully fabricated from periosteum from the mini-pig cranium. In vitro experiments indicated that the DP scaffold modulated macrophage polarization toward a pro-regenerative M2 phenotype, which in turn facilitated migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. A rat GBR model with a cranial critical-size defect was established, and our in vivo experiment confirmed the beneficial effects of DP on the local immune microenvironment and bone regeneration. Collectively, the findings of this study indicate that the prepared DP possesses immunomodulatory properties and represents a promising barrier membrane for GBR procedures.

A document by Miaomiao Wang. Click on the document to view its contents.

The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be carried out at medium cell density without effects on cell-specific titer or particle functionality, resulting in titers above 1010 VG/mL. Medium supplementation alleviated the cell density effect, in terms of VG/cell, at high-cell-density productions. On the other hand, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations. The medium-cell-density production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.

Adipose-derived stem cells (ADSCs) have important applications in basic research, especially in fat transplantation. Some studies have found that three-dimensional (3D) spheroids formed by mesenchymal stem cells have enhanced therapeutic potential. However, the fundamental basics of this effect are still being discussed. In this study, ADSCs were harvested from subcutaneous adipose tissues and 3D spheroids were formed by the automatic aggregation of ADSCs in a non-adhesive 6-well plate. Oxygen glucose deprivation (OGD) was used to simulate the transplantation microenvironment. We found that 3D culture of ADSCs triggered cell autophagy. After inhibiting autophagy by Chloroquine, the rates of apoptosis were increased. When the 3D ADSC-spheroids were re-planked, the number of senescent ADSCs decreased, and the proliferation ability was promoted. In addition, there were more cytokines secreted by 3D ADSC-spheroids including VEGF, IGF-1 and TGF-β. After adding the conditioned medium with human umbilical vein endothelial cells (HUVECs), 3D ADSC-spheroids were more likely to promote migration, and tube formation, stimulating the formation of new blood vessels. Fat grafting experiments in nude mice also showed that 3D ADSC-spheroids enhanced survival and neovascularization of fat grafts. These results suggested that 3D spheroids culturing of ADSCs can increase the therapeutic potential in fat transplantation.