Since the intestinal microbiota has a significant impact on the physiological state of the host, the development of technologies to control it is an urgent task. It is also thought that there are twice as many phages as bacteria in the intestine, and although the relationship between diseases and the intestinal phage flora is being reported, the overall structure of the intestinal phage flora and its relationship with diseases are still largely unknown. In this study, we performed metagenomic analysis of the intestinal phage flora of healthy Japanese people and multiple sclerosis (MS) patients to construct full-length genomes and deepen our understanding of the overall structure, and to explore the relationship between the intestinal phage flora and diseases and the possibility of controlling their pathology. Feces were collected from a total of 90 volunteers, including those with relapsing-remitting MS, progressive MS, and healthy individuals, and DNA and RNA were extracted, followed by shotgun metagenomics and RNA sequencing using short- and long-type sequencers. The resulting reads were used for assembly, quality checks, and high-quality contigs were obtained. The high-quality contigs were classified using various tools to obtain full-length or nearly full-length phage genomes. The obtained phage genomes were subjected to gene function analysis and host estimation to identify the characteristics of the phages and to confirm the relationship between gene expression levels and diseases. By assembling short and long reads, 6,185 nearly full-length phage genomes were obtained. When the obtained phage genomes were subjected to a homology search in existing phage genome databases, more than half were phages with novel genome sequences that had not been reported before. As a result of performing a pipeline process for host estimation, it was possible to estimate the hosts for approximately half of the phages. By using these phages as a reference, we analyzed the intestinal phage flora of MS patients and discovered phages and phage-derived functions (coenzyme B12 biosynthesis) that were significantly decreased by the disease. We also discovered active lysogenic phages that activate some pathogenic intestinal bacterial species by treating them with mitomycin. We are also investigating the control of pathogenic bacteria using these phages.

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In a thermophilic bacterium, Thermus thermophilus, lysine was found to be biosynthesized using a novel carrier protein (amino-group carrier protein: AmCP). It has been becoming clear that AmCP system is used for the biosynthesis of non-proteinogenic amino acids in various bacteria, including actinomycetes. In this study, we searched for novel natural products synthesized from non-proteinogenic amino acids that are biosynthesized through the AmCP system, and analyzed the biosynthetic pathways and mechanisms to expand the structural diversity of natural products.

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Anaerobic respiration using elemental sulfur as the terminal electron acceptor is a crucial metabolic process in sulfur-reducing bacteria and some archaea, playing an indispensable role in the biogeochemical sulfur cycle. This study aimed to elucidate the molecular mechanism of sulfur respiration in Geobacter sulfurreducens PCA. The protein encoded by the esuD gene was identified as a novel polysulfide reductase, MccSep, containing five c-type hemes and one Sec residue. The X-ray crystal structure of MccSep solved at 1.90Å revealed that Cys239 and Sec325, positioned near the His/Cys-coordinated heme, are crucial for the catalytic activity. The reduction of elemental sulfur and the growth of the ΔesuD strain on elemental sulfur media were significantly impaired compared to the parent strain, highlighting the crucial role of MccSep in sulfur respiration. The esuD gene was part of the esu operon, which includes esuABCDEFGHIJ on the G. sulfurreducens PCA genome. The lack of halo formation on the elemental sulfur agar medium by the ΔesuA, ΔesuB, ΔesuC, and ΔesuEFGHIJ strains showed the involvement of the operon in sulfur respiration. Expression of the operon genes was upregulated in response to elemental sulfur in the medium. Enzymatic analysis identified EsuA as a sulfur transferase with high catalytic efficiency for polysulfides using Cys330 as the catalytic residue. The ΔesuB strain expressing the plasmid-derived C147A mutant of EsuB did not complement sulfur respiration ability, demonstrating the critical role of Cys147 in EsuB for sulfur respiration. Similarly, introducing a plasmid expressing the ΔC104 mutant of EsuC into the ΔesuC strain did not restore sulfur respiration, indicating that Cys104 of EsuC is also essential for this process. Subcellular localization analysis showed that EsuA predominantly localized to the outer and inner membrane fractions, EsuB to the outer membrane, and ExtJ to the periplasm. These findings offer valuable insights into the molecular mechanisms underlying bacterial sulfur respiration.

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Unveiling biomedical functions of tumor-resident microbiota is challenging for developing advanced anticancer medicines. This study demonstrates that isolated intratumoral bacteria, associated with natural purple photosynthetic bacteria, have inherent biocompatibility and strong immunogenic anticancer efficacies. They preferentially grow and proliferate within a targeted tumor milieu, which effectively causes immune cells to infiltrate the tumor and provoke strong anticancer responses in various syngeneic mouse models, including colorectal cancer, sarcoma, metastatic lung cancer, and extensive drug-resistant breast cancer. Furthermore, these functional bacteria-treated mice exhibit excellent anticancerous responses and have significantly prolonged survival rates with effective immunological memory. Light-harvesting nanocomplexes of microbial consortia of intratumoral bacteria and purple photosynthetic bacteria can diagnose tumors using bio-optical-window near-infrared light, making them useful theranostic agents for highly targeted immunological elimination of the tumor and for precisely marking tumor location.

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Despite the fact that membrane transport proteins are indispensable for biological activities, they are not well understood due to the lack of sufficient research methods. Membrane transport proteins function under the influence of surrounding lipid molecules and membrane potential with large conformational changes. However, due to the above limitations in research methods, the understanding of the interaction of membrane transport proteins with lipid molecules and their operating mechanisms based on their dynamic behavior is not sufficient. Therefore, although membrane transport is very important in developing microbial cell factories (MCF), there is a lack of understanding especially of exporter proteins, which is one of the bottlenecks in MCF development. Membrane transport also plays an important role in major energy conversion processes in living organisms, such as respiration and photosynthesis. For example, several ion transport proteins that are expressed on thylakoid membranes and involved in photosynthetic activity have been identified. However, some ion transport proteins remain to be identified, while their existence has been suggested. We aimed to overcome the limitations of research methods in the study of membrane transport proteins by utilizing innovative analytical techniques developed by our own research group, as are below. (1)Original membrane transport analysis system that enables patch clamp analysis of microorganisms and organelles (2)High-precision molecular dynamics simulation of membrane transport proteins using originally developed force field Using analytical methods described above, we have been tackling the following research agenda in basic research on membrane transport proteins for exporter engineering and other applications towards sustainable society as below. (1)Analysis of mechanosensitive channels and their application to microbial membrane transport engineering, (2)Identification of the 5′-IMP exporter protein gene of Corynebacterium stationis and elucidation of the mechanism of improving the exporter protein activity, (3)Elucidation of the dynamic behavior of membrane transport proteins and analysis of their operating mechanisms using high-precision all-atom molecular dynamics simulation, (4)Electrophysiological analysis of thylakoid membrane-localized ion channels and their physiological roles. As a result, we have achieved some unique and valuable results.

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Although synthetic biology has made remarkable progress in recent years toward the design of microbial cell factories (MCFs) that produce a variety of valuable channels, many unexplored areas in the field of exporter engineering still remain due to the difficulty of research. We focused on mechanosensitive channels (Msc), which are responsible for L-Glu export in industrial L-Glu-producing MCF. In this study, we characterized the properties of Msc and explored its potential as a versatile exporter. The introduction of Msc genes carrying a gain-of-function (GOF) mutation into MCFs successfully increased the production rate of valuable chemicals such as L-Lys, and inosine 5′-monophosphate (5′-IMP). In contrast to the conventional view that high specificity is required for an exporter, our results demonstrated that even a channel with low selectivity can function effectively as an exporter in MCF. Furthermore, our findings on the dynamic gating behavior of mechanosensitive channel of large conductance (MscL) obtained by all-atom molecular dynamics simulations provide clues to understanding the mechanism by which G46D substitution of MscL leads to GOF. Specifically, the G46D substitution causes a kink at A38 of the first transmembrane helix (TM1), which may affect the interaction between TM1 and TM2 and destabilize the gate in closure state, resulting in a GOF. To verify this, we introduced a double substitution gene with A38V and G46D, which interferes with A38 kink, into 5′-IMP-producing MCF, and the effect of G46D on 5′-IMP production rate per cell was abolished. These demonstrate that molecular dynamics simulation is effective in understanding the complex mechanism of the Msc gating and has great potential as a tool for designing functionally improved Msc.

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Product secretion from an engineered cell can be advantageous for microbial cell factories. Extensive work on nucleotide manufacturing, one of the most successful microbial fermentation processes, has enabled Corynebacterium stationis to transport nucleotides outside the cell by random mutagenesis; however, the underlying mechanism has not been elucidated, hindering its applications in transporter engineering. Herein, we report the nucleotide-exporting major facilitator superfamily (MFS) transporter from the C. stationis genome and its hyperactive mutation at the G64 residue. Structural estimation and molecular dynamics simulations suggested that the activity of this transporter improved via two mechanisms: (1) enhancing interactions between transmembrane helices through the conserved “RxxQG” motif along with substrate binding and (2) trapping substrate-interacting residue for easier release from the cavity. Our results provide novel insights into how MFS transporters change their conformation from inward- to outward-facing states upon substrate binding to facilitate export and can contribute of rational design strategies for improvement of exporters in microbial cell factories.

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This study focuses on analyzing the function of membrane transport proteins using high-precision all-atom molecular dynamics (MD) simulations, examining three key proteins: the sodium/proton antiporter A (NhaA), the light-driven proton pump bacteriorhodopsin, and the multidrug and toxic compound extrusion (MATE) transporter. For NhaA, it was found that the membrane of the cyclopropane fatty acid synthase gene disruptant showed tighter lipid packing and stronger interactions with NhaA compared to wild-type membranes, resulting in significant conformational changes in transmembrane helix 5 and enhanced Na transport activity. In the study of bacteriorhodopsin, new lipid force fields for archaeal membranes were developed, revealing that light activation strengthens interactions between retinal and key amino acids (Asp212 and Asp85) and highlighting the role of water molecule dynamics in proton transport. Differences in interactions between bacteriorhodopsin and archaeal membranes versus phosphatidylethanolamine (PE) membranes were also explored, showing significant structural impacts due to lipid composition. In the study of the MATE transporter, systems with varying protonation states of conserved acidic residues (Asp36, Glu255, Asp371) were examined, revealing that these residues play crucial roles in ion binding and transport. In particular, it was suggested that the protonation of Asp371 facilitates sodium uptake in the outward-facing state and supports sodium trapping by Glu255 in the inward-facing state. These findings provide detailed insights into the dynamic behavior and interaction mechanisms of membrane transport proteins, highlighting the importance of lipid composition and conserved residues in their function, contributing to a deeper understanding of membrane protein behavior, and informing drug development, biotechnological applications, and microbial-based production.

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Studies on transporters that localize to the thylakoid membrane and are involved in regulating photosynthetic activity have been limited to transport proteins in Arabidopsis thaliana. These transporters have all been shown to regulate photosynthetic activity under fluctuating light conditions. However, there are very few reports of transport proteins involved in photosynthetic activity in cyanobacteria. In this study, we focused on the functional analysis of the ion channel Stc1, which localizes to the thylakoid membrane of the cyanobacteria Synechocystis sp. PCC 6803. The ∆stc1 strain showed retarded growth and significantly reduced photosynthetic activity under low concentrations of K⁺, suggesting that it causes abnormalities in electron transfer after PSII in the photosynthetic electron-transfer chain. Furthermore, electrophysiological analysis using patch-clamp assay in E. coli mutants expressing stc1 revealed its ion transport activity. This study clarified the detailed transport activity and physiological role of Stc1 The photosynthetic activity of the ∆stc1 strain was significantly reduced. So far, no ion transport protein, except H⁺ translocating proteins, has been reported to play such an important role in photosynthetic activity in plant and cyanobacterial photosynthesis studies. In the long history of photosynthesis research, the protein molecules that appear in photosynthetic reactions seemed to be almost completely clarified, however, Stc1 was shown to be a new molecule to add to the list.

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