Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system.

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

Engineered chlorophyll catabolism conferring predator resistance for microalgal biomass production.

Production of valuable compounds including biofuels and pharmaceutical precursors derived from microalgae has garnered significant interest. Stable production of algal biomass is essential to make the microalgal industry commercially feasible. However, one of the largest issues is severe biological contamination by predators grazing the algal biomass, resulting in the crash of outdoor cultures. In the present study, we propose a novel engineering strategy for microalgae to cope with predators. The overexpression of plant chlorophyllase (CLH) in a microalga resulted in the enhancement of resistance to the predator. This result supported our hypothesis that CLH promotes chlorophyll breakdown in the chloroplasts of the microalgae when they are digested by the predator, generating the phototoxic catabolite chlorophyllide that damages the predator. To the best of our knowledge, this is the first study to establish predator-resistant microalgae by enhancing the CLH activity.

Prostaglandin production by the microalga with heterologous expression of cyclooxygenase.

Prostaglandins (PGs) are the physiologically active compounds synthesized from C20 polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) and a series of PG synthases, and are utilized as pharmaceuticals. Currently, commercialized PGs are mainly produced by chemical synthesis under harsh conditions. By contrast, bioproduction of PGs can be an alternative, environmental-friendly, and inexpensive process with genetic engineering of model plants, although these conventional host organisms contain a limited quantity of PG precursors. In this study, we established an efficient PG production process using the genetically engineered microalga Fistulifera solaris which is rich in C20 PUFAs. A cox gene derived from the red alga Agarophyton vermiculophyllum was introduced into F. solaris. As a result, a transformant clone with high cox expression produced PGs (i.e., PGD2 , PGE2 , PGF2α , and 15-ketoPGF2α derived from arachidonic acid, and PGD3 , PGE3 , and PGF3α derived from eicosapentaenoic acid) as revealed by liquid chromatography/mass spectrometry. The total content of PGs was 1290.4 ng/g of dry cell weight, which was higher than that produced in the transgenic plant reported previously. The results obtained in this study indicate that the C20 PUFA-rich microalga functionally expressing COX is a promising host for PG bioproduction.

Lensless imaging-based discrimination between tumour cells and blood cells towards circulating tumour cell cultivation.

Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.

Gel-based cell manipulation method for isolation and genotyping of single-adherent cells.

Genetic analysis of single-cells is widely recognized as a powerful tool for understanding cellular heterogeneity and obtaining genetic information from rare populations. Recently, many kinds of single-cell isolation systems have been developed to facilitate single-cell genetic analysis. However, these systems mainly target non-adherent cells or cells in a cell suspension. Thus, it is still challenging to isolate single-adherent cells of interest from a culture dish using a microscope. We had previously developed a single-cell isolation technique termed "gel-based cell manipulation" (GCM). In GCM, single-cells could be visualized by photopolymerizable-hydrogel encapsulation that made it easier to isolate the single-cells. In this study, GCM-based isolation of single-adherent cancer cells from a culture dish was demonstrated. Single-adherent cells were encapsulated in a photopolymerizable hydrogel using a microscope and isolated with high efficiency. Furthermore, whole genome amplification and sequencing for the isolated single-adherent cell could be achieved. We propose that the GCM-based approach demonstrated in this study has the potential for efficient analysis of single-adherent cells at the genetic level.

Homoeolog expression bias in allopolyploid oleaginous marine diatom Fistulifera solaris.

BACKGROUND: Allopolyploidy is a genomic structure wherein two or more sets of chromosomes derived from divergent parental species coexist within an organism. It is a prevalent genomic configuration in plants, as an important source of genetic variation, and also frequently confers environmental adaptability and increased crop productivity. We previously reported the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to be a promising host for biofuel production and that its genome is allopolyploid, which had never previously been reported in eukaryotic microalgae. However, the study of allopolyploidy in F. solaris was hindered by the difficulty in classifying the homoeologous genes based on their progenitor origins, owing to the shortage of diatom genomic references. RESULTS: In this study, the allopolyploid genome of F. solaris was tentatively classified into two pseudo-parental subgenomes using sequence analysis based on GC content and codon frequency in each homoeologous gene pair. This approach clearly separated the genome into two distinct fractions, subgenome Fso_h and Fso_l, which also showed the potency of codon usage analysis to differentiate the allopolyploid subgenome. Subsequent homoeolog expression bias analysis revealed that, although both subgenomes appear to contribute to global transcription, there were subgenomic preferences in approximately 61% of homoeologous gene pairs, and the majority of these genes showed continuous bias towards a specific subgenome during lipid accumulation. Additional promoter analysis indicated the possibility of promoter motifs involved in biased transcription of homoeologous genes. Among these subgenomic preferences, genes involved in lipid metabolic pathways showed interesting patterns in that biosynthetic and degradative pathways showed opposite subgenomic preferences, suggesting the possibility that the oleaginous characteristics of F. solaris derived from one of its progenitors. CONCLUSIONS: We report the detailed genomic structure and expression patterns in the allopolyploid eukaryotic microalga F. solaris. The allele-specific patterns reported may contribute to the oleaginous characteristics of F. solaris and also suggest the robust oleaginous characteristics of one of its progenitors. Our data reveal novel aspects of allopolyploidy in a diatom that is not only important for evolutionary studies but may also be advantageous for biofuel production in microalgae.

High-Throughput Manipulation of Circulating Tumor Cells Using a Multiple Single-Cell Encapsulation System with a Digital Micromirror Device.

Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms.

Biosynthesis of Thermoresponsive Magnetic Nanoparticles by Magnetosome Display System.

Thermoresponsive magnetic nanoparticles (MNPs) were synthesized using a magnetosome display system. An elastin-like polypeptide decamer of VPGVG (ELP10), which is hydrophobic above the transition temperature ( Tt) and can form an insoluble aggregation, was immobilized on biogenic MNPs in the magnetotactic bacterium, Magnetospirillum magneticum AMB-1. It was suggested that hydrophobicity of the MNP surface increased at 60 °C compared with 20 °C by the immobilization of ELP10. Size distribution analysis indicated that the immobilization of ELP10 onto MNPs induced the increased hydrophobicity with increasing temperatures up to 60 °C, promoting aggregation of the particles by hydrophobic and magnetic interactions. These results suggest that the acceleration of magnetic collection at 60 °C was caused by particle aggregation promoted by hydrophobic interaction between ELP-MNPs. Furthermore, the immobilization of ELP on MNPs gave a quick magnetic collection at 60 °C by external magnetic field. The thermoresponsive properties will further expand the utility of biotechnological applications of biogenic MNPs.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Colony Fingerprint-Based Discrimination of Staphylococcus species with Machine Learning Approaches.

Detection and discrimination of bacteria are crucial in a wide range of industries, including clinical testing, and food and beverage production. Staphylococcus species cause various diseases, and are frequently detected in clinical specimens and food products. In particular, S. aureus is well known to be the most pathogenic species. Conventional phenotypic and genotypic methods for discrimination of Staphylococcus spp. are time-consuming and labor-intensive. To address this issue, in the present study, we applied a novel discrimination methodology called colony fingerprinting. Colony fingerprinting discriminates bacterial species based on the multivariate analysis of the images of microcolonies (referred to as colony fingerprints) with a size of up to 250 µm in diameter. The colony fingerprints were obtained via a lens-less imaging system. Profiling of the colony fingerprints of five Staphylococcus spp. (S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. simulans) revealed that the central regions of the colony fingerprints showed species-specific patterns. We developed 14 discriminative parameters, some of which highlight the features of the central regions, and analyzed them by several machine learning approaches. As a result, artificial neural network (ANN), support vector machine (SVM), and random forest (RF) showed high performance for discrimination of theses bacteria. Bacterial discrimination by colony fingerprinting can be performed within 11 h, on average, and therefore can cut discrimination time in half compared to conventional methods. Moreover, we also successfully demonstrated discrimination of S. aureus in a mixed culture with Pseudomonas aeruginosa. These results suggest that colony fingerprinting is useful for discrimination of Staphylococcus spp.

Evaluation of cancer cell deformability by microcavity array.

A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 µm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy.

Production of ω3 fatty acids in marine cyanobacterium Synechococcus sp. strain NKBG 15041c via genetic engineering.

Omega-3 fatty acids (ω3 FAs) have attracted attention because they have various health benefits for humans. Fish oils are currently major sources of ω3 FAs, but a sustainable supply of ω3 FAs based on fish oils is problematic because of the increasing demand. In this study, the production potential of a genetically engineered marine cyanobacterium, Synechococcus sp. strain NKBG 15041c, was examined as an alternative source of ω3 FAs. A change in fatty acid composition of this cyanobacterium was successfully induced by the expression of a heterologous Δ6-desaturase, and the transformants synthesized stearidonic acid, which the wild type cannot produce. As a result of optimization of culture conditions, maximal contents of stearidonic acid and total ω3 FAs reached 12.2 ± 2.4 and 118.1 ± 3.5 mg/g, respectively. The maximal ω3 FA productivity was 4.6 ± 0.7 mg/(L⋅day). These are the highest values of the contents of stearidonic acid and ω3 FAs in genetically engineered cyanobacteria reported thus far. Therefore, genetically engineered Synechococcus sp. strain NKBG 15041c may be a promising sustainable source of ω3 fatty acids.

Outdoor Cultivation of Marine Diatoms for Year-Round Production of Biofuels.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April-October) and Mayamaea sp. JPCC CTDA0820 (November-March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m² day) for F. solaris, and 8.62 and 0.92 g/(m² day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.

Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

Manipulation of a Single Circulating Tumor Cell Using Visualization of Hydrogel Encapsulation toward Single-Cell Whole-Genome Amplification.

Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs.

Functional expression of full-length TrkA in the prokaryotic host Magnetospirillum magneticum AMB-1 by using a magnetosome display system.

Tropomyosin receptor kinase A (TrkA), a receptor tyrosine kinase, is known to be associated with various diseases. Thus, TrkA has become a major drug-screening target for these diseases. Despite the fact that the production of recombinant proteins by prokaryotic hosts has advantages, such as fast growth and ease of genetic engineering, the efficient production of functional receptor tyrosine kinase by prokaryotic hosts remains a major experimental challenge. Here, we report the functional expression of full-length TrkA on magnetosomes in Magnetospirillum magneticum AMB-1 by using a magnetosome display system. TrkAwas fused with the magnetosome-localized protein Mms13 and expressed on magnetosome surfaces. Recombinant TrkA showed both nerve growth factor (NGF)-binding and autophosphorylation activities. TrkA expressed on magnetosomes has the potential to be used, not only for further functional analysis of TrkA, but also for ligand screening.

Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis.

The immobilization of multiple cellulase complexes receiving attention for use in the efficient hydrolysis of celluloses. In this study, the magnetosome display system was employed for the preparation of systems mimicking natural multiple cellulase complexes (cellulosomes) on magnetic nanoparticles (MNPs). Initially, two fluorescent proteins, namely, green fluorescent protein and mCherry, were immobilized on MNPs. Fluorescence analysis revealed the close proximity of two different proteins on the MNPs. Enzyme-linked immunosorbent assay analysis showed that stoichiometrically equivalent amounts of the proteins were immobilized on the MNPs. Next, endoglucanase (EG) and ß-glucosidase (BG) were immobilized on MNPs to give EG/BG-MNPs. The resulting MNPs were applied for the hydrolysis of celluloses, with rapid hydrolysis of carboxymethyl cellulose being observed. Furthermore, the fusion of the cellulose-binding domain to EG/BG-MNPs promoted improved hydrolysis activity against the insoluble cellulose. We could therefore conclude that the magnetosome display system can expand the possibilities of mimicking natural cellulosome organization on MNPs.

Alkane production by the marine cyanobacterium Synechococcus sp. NKBG15041c possessing the α-olefin biosynthesis pathway.

The production of alkanes in a marine cyanobacterium possessing the α-olefin biosynthesis pathway was achieved by introducing an exogenous alkane biosynthesis pathway. Cyanobacterial hydrocarbons are synthesized via two separate pathways: the acyl-acyl carrier protein (ACP) reductase/aldehyde-deformylating oxygenase (AAR/ADO) pathway for the alkane biosynthesis and the α-olefin synthase (OLS) pathway for the α-olefin biosynthesis. Coexistence of these pathways has not yet been reported. In this study, the marine cyanobacterium Synechococcus sp. NKBG15041c was shown to produce α-olefins similar to those of Synechococcus sp. PCC7002 via the α-olefin biosynthesis pathway. The production of heptadecane in Synechococcus sp. NKBG15041c was achieved by expressing the AAR/ADO pathway genes from Synechococcus elongatus PCC 7942. The production yields of heptadecane in Synechococcus sp. NKBG15041c varied with the expression level of the aar and ado genes. The maximal yield of heptadecane was 4.2 ± 1.2 µg/g of dried cell weight in the transformant carrying a homologous promoter. Our results also suggested that the effective activation of ADO may be more important for the enhancement of alkane production by cyanobacteria.

In vivo live cell imaging for the quantitative monitoring of lipids by using Raman microspectroscopy.

A straightforward in vivo monitoring technique for biomolecules would be an advantageous approach for understanding their spatiotemporal dynamics in living cells. However, the lack of adequate probes has hampered the quantitative determination of the chemical composition and metabolomics of cellular lipids at single-cell resolution. Here, we describe a method for the rapid, direct, and quantitative determination of lipid molecules from living cells using single-cell Raman imaging. In vivo localization of lipids in the form of triacylglycerol (TAG) within oleaginous microalga and their molecular compositions are monitored with high spatial resolution in a nondestructive and label-free manner. This method can provide quantitative and real-time information on compositions, chain lengths, and degree of unsaturation of fatty acids in living cells for improving the cultivating parameters or for determining the harvest timing during large-scale cultivations for microalgal lipid accumulation toward biodiesel production. Therefore, this technique is a potential tool for in vivo lipidomics for understanding the dynamics of lipid metabolisms in various organisms.