Genetically engineered bacteriophages as novel nanomaterials: applications beyond antimicrobial agents.

Bacteriophages, also known as phages, are viruses that replicate in bacteria and archaea. Phages were initially discovered as antimicrobial agents, and they have been used as therapeutic agents for bacterial infection in a process known as "phage therapy." Recently, phages have been investigated as functional nanomaterials in a variety of areas, as they can function not only as therapeutic agents but also as biosensors and tissue regenerative materials. Phages are nontoxic to humans, and they possess self-assembled nanostructures and functional properties. Additionally, phages can be easily genetically modified to display specific peptides or to screen for functional peptides via phage display. Here, we demonstrated the application of phage nanomaterials in the context of tissue engineering, sensing, and probing.

A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT-PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.

Virus-like nanoparticles as a theranostic platform for cancer.

Virus-like nanoparticles (VLPs) are natural polymer-based nanomaterials that mimic viral structures through the hierarchical assembly of viral coat proteins, while lacking viral genomes. VLPs have received enormous attention in a wide range of nanotechnology-based medical diagnostics and therapies, including cancer therapy, imaging, and theranostics. VLPs are biocompatible and biodegradable and have a uniform structure and controllable assembly. They can encapsulate a wide range of therapeutic and diagnostic agents, and can be genetically or chemically modified. These properties have led to sophisticated multifunctional theranostic platforms. This article reviews the current progress in developing and applying engineered VLPs for molecular imaging, drug delivery, and multifunctional theranostics in cancer research.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.

Enzyme immobilization on porous chitosan hydrogel capsules formed by anionic surfactant gelation.

OBJECTIVES: Sodium dodecyl sulfate (SDS)-chitosan hydrogels have been employed for adsorption of anionic dyes and metallic substances. Two mutant forms of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) were used as model enzymes to develop a novel enzyme immobilization technique employing newly formulated porous chitosan hydrogels. RESULTS: The enzyme immobilized on chitosan hydrogel capsules formed by 5 g/l SDS gelation and subsequent treatment with 0.05 M NaOH was 28-35% higher in NADPH production than that formed by 20 g/l SDS gelation only under the same conditions. A 48-h asymmetric biphasic reduction of acetophenone with immobilized TeSADH enzyme at 50 °C showed 68% increase in (R)-1-phenylethanol production than the free enzyme. Compared to the free enzyme which denatured and lost its activity at 80 °C, the immobilized enzyme retained about 25% of its initial activity after 2-h incubation. CONCLUSION: In contrast to the conventional chitosan hydrogel which suffers thermal and operational stability, the newly formulated porous chitosan hydrogel capsules have excellent enzyme loading efficiency and stable at harsh temperatures. Especially, this newly developed enzyme immobilization method would be applicable for food processing.

Coexpression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells.

Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5'-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.

Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase.

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (kcat/KM) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'- methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/ C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °C with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'- methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.

On-chip biosynthesis of GM1 pentasaccharide-related complex glycans.

A functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and to apply glycan-involved applications simultaneously. GM3 trisaccharide, GM2 tetrasaccharide, and GM1 pentasaccharide were successfully directly biosynthesized on lactose-immobilized surfaces through three consecutive glycosyltransferase reactions along with small amounts of enzymes and donors, without any additional processes. Biosynthesized GM1 pentasaccharide-related complex glycans were demonstrated to provide information on the substrate specificity of whole cholera toxin. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan-involved applications such as glycan-protein interaction analysis and glycan biomarker-based diagnosis.

Diatom-Inspired Silica Nanostructure Coatings with Controllable Microroughness Using an Engineered Mussel Protein Glue to Accelerate Bone Growth on Titanium-Based Implants.

Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium-based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein-based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica-precipitating R5 peptide (R5-MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness-dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5-MAP-based SiNP nanostructure assembly could be practically applied to accelerate bone-tissue growth to improve the stability and prolong the lifetime of medical implantable devices.

Mutagenesis of Met-151 and Thr-153 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase changes substrate specificity for acetophenones.

Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.

I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones.

Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.; Phillips, R. S. ChemCatChem2009, 1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/C295A SADH has broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates.

Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously.

Mechanically Durable and Biologically Favorable Protein Hydrogel Based on Elastic Silklike Protein Derived from Sea Anemone.

As biodegradable scaffolds, protein hydrogels have considerable potential, particularly for bioartificial organs and three-dimensional space-filling materials. However, their low strength and stiffness have been considered to be limitations for enduring physiological stimuli. Therefore, protein hydrogels have been commonly utilized as delivery vehicles rather than as supporting materials. In this work, sea anemone tentacle-derived recombinant silk-like protein (aneroin) was evaluated as a potential material for a mechanically durable protein hydrogel. Inspired by the natural hardening mechanism, photoinitiated dityrosine cross-linking was employed to fabricate an aneroin hydrogel. It was determined that the fabricated aneroin hydrogel was approximately 10-fold stiffer than mammalian cardiac or skeletal muscle. The aneroin hydrogel provided not only structural support but also an adequate environment for cells. It exhibited an adequate swelling ability and microstructure, which are beneficial for facilitating mass transport and cell proliferation. Based on its mechanical and biological properties, this aneroin hydrogel could be used in various biomedical applications, such as cell-containing patches, biomolecule carriers, and artificial extracellular matrices.

Optical detection of paraoxon using single-walled carbon nanotube films with attached organophosphorus hydrolase-expressed Escherichia coli.

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

A nonfusogenic antigen mimic of influenza hemagglutinin glycoproteins constituted with soluble full-length HA1 and truncated HA2 proteins expressed in E. coli.

A novel method is proposed to produce a soluble recombinant antigen mimic, constituted with full-length HA1 and truncated HA2 individually expressed in E. coli, instead of a precursor form of hemagglutinin protein, that is similar to the naturally processed and disulfide-linked HA1/HA2 on the envelope of the influenza A virus strain X-31 (H3N2). A truncated ectodomain of HA2 subunit, HA2(23-185)/C137S, lacked two membrane-interacting sequences, i.e., the N-terminal fusion peptide as well as the transmembrane domain and short cytoplasmic segment at the C terminus. A recombinant HA1 (rHA1) subunit protein, HA1(1-328)/C14S/L157S, lacked the signal peptide. Mutations C137S and C14S in the HA2 and HA1 subunits, respectively, were introduced to prevent any possible disulfide linkage between the two subunit proteins. The rHA antigen mimic would be nonfusogenic mainly due to the absence of the N-terminal fusion peptide as well as the C-terminal transmembrane domain in the truncated HA2, and eventually less cytotoxic as well. Antibody responses induced by two soluble rHA antigens were evaluated by ELISA assays to detect rHA antigens injected and to validate both anti-HA1 and anti-HA2 antibodies produced in the mice sera. Antigenic rHA proteins also elicited neutralizing antibodies against homologous H3N2 influenza virus in the immunized mice, without severe body weight loss or any other adverse symptoms.

Rg3-enriched Korean Red Ginseng improves vascular function in spontaneously hypertensive rats.

BACKGROUND: Panax ginseng has distinct and impressive health benefits, such as improved blood pressure and immune system functioning. Rg3-enriched Korean Red Ginseng (REKRG) isolated from Korean Red Ginseng contains a high percentage of Rg3. METHODS: In this study, we examined the effects of REKRG on endothelial cell nitric oxide synthase (eNOS) activation and adhesion molecules in endothelial cells and vascular function in rats. RESULTS: REKRG dose-dependently increased eNOS phosphorylation and nitric oxide (NO) production in endothelial cells. In addition, REKRG markedly inhibited the tumor necrosis factor-α (TNF-α)-mediated induction of intercellular adhesion molecule (ICAM)-1 and cyclooxygenase (COX)-2 expressions in endothelial cells. REKRG improved endothelium-dependent vasorelaxation in the Wistar-Kyoto (WKY) rat and spontaneously hypertensive rats (SHRs) compared with controls. Furthermore, REKRG treatment for 6 weeks increased serum NO levels and reduced the mean aortic intima-media thickness compared with controls. CONCLUSION: Taken together, these results suggest that REKRG increased vascular function and improved immune system functioning. Therefore, REKRG is a very useful food for preventing or improving various cardiovascular diseases.

Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds.

The development of efficient tools is required for the eco-friendly detoxification and effective detection of neurotoxic organophosphates (OPs). Although enzymes have received significant attention as biocatalysts because of their high specific activity, the uneconomic and labor-intensive processes of enzyme production and purification make their broad use in practical applications difficult. Because whole-cell systems offer several advantages compared with free enzymes, including high stability, a reduced purification requirement, and low preparation cost, they have been suggested as promising biocatalysts for the detoxification and detection of OPs. To develop efficient whole-cell biocatalysts with enhanced activity and a broad spectrum of substrate specificity, several factors have been considered, namely the selected strains, the chosen OP-hydrolyzing enzymes, where enzymes are localized in a cell, and which enhancer will assist the expression, function, and folding of the enzyme. In this article, we review the current investigative progress in the development of engineered whole-cell biocatalysts with excellent OP-hydrolyzing activity, a broad spectrum of substrate specificity, and outstanding stability for the detoxification and detection of OPs.