{"title":"Mechanosensitive channels are versatile exporters in microbial cell factories.","authors":"Tomoyuki Konishi, Yasuyuki Sawada, Ken-Ichi Hashimoto, Isamu Yabe, Masahiro Sokabe, Hisashi Kawasaki","doi":"10.2323/jgam.2025.09.002","DOIUrl":"https://doi.org/10.2323/jgam.2025.09.002","url":null,"abstract":"<p><p>The extracellular export of target chemicals is essential for achieving the target productivity of microbial cell factories (MCFs). We demonstrated that MscCG, a mechanosensitive channel responsible for glutamate export in glutamate-producing MCF of Corynebaterium glutamicum, can export various intracellular low-molecular-weight chemicals outside the cell. The mechanosensitive channels exporter improved L-Lys productivity and conferred substantial 5'-IMP fermentative production ability to the Escherichia coli MCF, which lacks inherent 5'-IMP exporters, indicating that mechanosensitive channels, which are low selective, functioned effectively as MCF exporters. We also demonstrated the effectiveness of a gain-of-function (GOF) mutation of mechanosensitive channels as MCF exporters; however, the essential mechanism underlying this GOF mutation remains unknown. Therefore, we performed molecular dynamics simulations to identify this mechanism at the atomic level. Consequently, we partially elucidated the underlying mechanism of G46D-induced GOF in MscL, which was effective as a 5'-IMP exporter. Specifically, the kink at A38 in the inner transmembrane helix of MscL forming its pore can affect GOF behavior. Based on these results, we conclude that mechanosensitive channels have potential as innovative and versatile exporters of MCFs, capable of enhancing the production efficiency of target chemicals and enabling their production in the absence of natural exporters.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145185956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterization of the acylglycerolphosphate acyltransferase in Actinoplanes missouriensis.","authors":"Shixuan Hu, Takeaki Tezuka, Yasuo Ohnishi","doi":"10.2323/jgam.2025.09.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.09.001","url":null,"abstract":"<p><p>Actinoplanes missouriensis grows by forming branched substrate mycelia and produces terminal sporangia. Each sporangium contains a few hundred spores, which swim as zoospores after being released from sporangia. Previously, we disrupted 22 putative acyltransferase genes and examined their involvement in morphological differentiation. Here, we described the characterization of one of them, a putative 1-acyl-2-hydroxy-sn-glycerol-3-phosphate acyltransferase (AGPAT) encoded by plsC (AMIS_11360). The plsC null (∆plsC) mutant exhibited a conditional growth defect in a nutrient-poor medium. No differences were observed between the wild-type and ∆plsC strains in sporangium formation, spore release, or zoospore motility. We confirmed the AGPAT activity of PlsC; the recombinant polyhistidine-tagged PlsC protein transferred the acyl group from palmitoyl-coenzyme A to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, resulting in the production of 1,2-dipalmitoyl-sn-glycero-3-phosphate.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145075396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Diverse fungal communities in commercial karebushi produced with or without a starter culture.","authors":"Kazuna Yanagi, Kentaro Hiramatsu, Chihiro Kadooka, Atsushi Nishitani, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Hisanori Tamaki, Taiki Futagami","doi":"10.2323/jgam.2025.08.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.08.001","url":null,"abstract":"<p><p>We investigated the effect of a starter culture on the fungal communities of commercial karebushi. Aspergillus pseudoglaucus was initially identified as the starter fungus. In karebushi samples from two manufacturers relying on naturally occurring molds, Aspergillus chevalieri was the dominant species, accompanied by Aspergillus montevidensis and Aspergillus sydowii, while A. pseudoglaucus was not detected. Among samples from six manufacturers that used the starter culture, A. pseudoglaucus was dominant in only three; in the remaining three, A. chevalieri predominated despite the starter being used. These results suggest that indigenous fungi, particularly A. chevalieri, present in the processing environment can outcompete the starter culture, influence the fungal community, and potentially contribute to the qualitative diversity of karebushi.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterization of the Azotobacter vinelandii nitrogenase complex expressed in Escherichia coli toward further activity improvement.","authors":"Yusuke Ito, Daisuke Yoshidome, Makoto Hidaka, Yasuko Araki, Kotaro Ito, Saori Kosono, Makoto Nishiyama","doi":"10.2323/jgam.2024.12.001","DOIUrl":"10.2323/jgam.2024.12.001","url":null,"abstract":"<p><p>We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD<sub>2</sub>K<sub>2</sub>, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD<sub>2</sub>K<sub>2</sub> component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD<sub>2</sub>K<sub>2</sub> formation, effectively increased nitrogenase activity.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142978944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"CRISPR-Cas9 genome editing of miso and soy source yeast Zygosaccharomyces sp.","authors":"Tomoo Ogata, Kotori Koide, Shiori Kudou, Miu Suto, Kotaro Uehara, Teruya Kaneko","doi":"10.2323/jgam.2025.04.002","DOIUrl":"10.2323/jgam.2025.04.002","url":null,"abstract":"<p><p>Genome modification would be useful for developing breeding techniques for haploid Zygosaccharomyces rouxii and natural hybrid allodiploid Zygosaccharomyces sp. yeast strains used in miso and soy sauce production. In this study, genome editing using CRISPR-Cas9 was attempted in Zygosaccharomyces sp. strains. Based on techniques in Saccharomyces cerevisiae, the Cas9 gene and guide RNA (gRNA) were expressed from the same plasmid. Targeting of the ZygoLEU2 gene of haploid Z. rouxii strain DA2 led to of a single-nucleotide insertion in the ORF, resulting in termination of translation at 10 amino acids. This single-base insertion was 3-bp upstream of the protospacer-associated motif (PAM) sequence, suggesting that it occurred during the repair process following the Cas9-induced double-strand break. The transformant was auxotrophic for leucine, verifying that genome editing using CRISPR-Cas9 had occurred. Application of the CRISPR-Cas9 system to allodiploid Zygosaccharomyces sp. strains, which have T- and P-subgenomes, resulted in transformants with base insertions or deletions upstream of the PAM sequence, or insertions of different subgenome sequences. Leucine-auxotrophic transformants were obtained in which the ORF of the ZygoLEU2 gene in both subgenomes were mutated. In some genome-edited strains, a significant region of one subgenome chromosome was missing. Lastly, we applied CRISPR-Cas9 to the gene encoding Hog1, a protein kinase involved in adaptation to high-salt and high-osmolarity conditions. Mutation of the HOG1 genes of both the T- and P-subgenomes by CRISPR-Cas9 significantly reduced growth in high salt and high osmolarity conditions.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Asmaa Ali Ahmed, Akiko Hida, Takahisa Tajima, Junichi Kato
{"title":"Identification and characterization of a methyl-accepting chemotaxis protein in Ralstonia pseudosolanacearum using chemically undefined materials.","authors":"Asmaa Ali Ahmed, Akiko Hida, Takahisa Tajima, Junichi Kato","doi":"10.2323/jgam.2025.03.001","DOIUrl":"10.2323/jgam.2025.03.001","url":null,"abstract":"<p><p>Ralstonia pseudosolanacearum is a plant-pathogenic bacterium that causes bacterial wilt in economically important crops. Chemotaxis is required for full virulence in R. pseudosolanacearum. R. pseudosolanacearum Ps29 possesses 20 methyl-accepting chemotaxis proteins (MCPs) and 2 MCP-like chemoreceptors. To understand the role of chemotaxis in plant infection, we are characterizing the functions of these 20 MCPs. Out of 20 MCPs, 8 MCPs have been characterized. To characterize the remaining MCPs, we deleted the 8 genes encoding characterized MCPs in R. pseudosolanacearum Ps29 to construct R. pseudosolanacearum D8. R. pseudosolanacearum D8 was examined for chemotactic responses to several chemically undefined materials including vegetable juices and tryptic soy broth (TSB) to find attractants. R. pseudosolanacearum D8 showed strong responses to green pepper and carrot juices and TSB. We constructed a mutant library of R. pseudosolanacearum D8 by deleting each of the MCP genes. Chemotaxis assays to TSB revealed that an MCP which we named McpD was responsible for sensing an attractant(s) in TSB. Because amino acids are the major constituents of TSB, we measured chemotactic responses of R. pseudosolanacearum D8 to 20 proteinogenic amino acids and found Asp and Glu as the major attractants of McpD and Cys as the minor attractant. R. pseudosolanacearum Ps29 can utilize Asp and Glu as sole carbon and nitrogen sources, suggesting that the role of McpD-mediated chemotaxis is finding growth substrates.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143639722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Screening to isolate Bacillus subtilis mutants with enhanced NADPH levels.","authors":"Yuzheng Wu, Shu Ishikawa, Ken-Ichi Yoshida","doi":"10.2323/jgam.2025.01.001","DOIUrl":"10.2323/jgam.2025.01.001","url":null,"abstract":"<p><p>As the first step toward understanding how NADPH levels are regulated in Bacillus subtilis, we sought to obtain mutant strains with enhanced NADPH levels. Our previous study demonstrated that in a strain of B. subtilis expressing bacterial luciferase derived from Photorhabdus luminescens, artificially enhancing NADPH levels enhanced luciferase luminescence in the colonies. In this study, from a library of ethyl methanesulfonate-treated mutants, those with enhanced luciferase luminescence in colonies were isolated, and five isolates were further selected by luminescence in microplate culture. Finally, we measured intracellular NADPH levels of them and found that all the five strains had significantly enhanced NADPH levels compared to the parental strain. In addition, four strains significantly increased total NADP(H) levels. These results demonstrate the effectiveness of our strategy as a methodology for obtaining mutant strains useful for elucidating the mechanisms for regulation of NADPH levels in B. subtilis.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Identification of Escherichia coli flagellar antigen by disc immuno-immobilization.","authors":"Shiho Oyama, Masatoshi Fujihara","doi":"10.2323/jgam.2025.04.001","DOIUrl":"10.2323/jgam.2025.04.001","url":null,"abstract":"<p><p>Disc immuno-immobilization is a simple method for typing flagellar antigens from Salmonella enterica. In this study, we successfully adapted this method for Escherichia coli. All eleven strains tested were determined their antigens within 14 h from inoculation. This method improves the efficiency and speed, highlighting its usefulness in clinical laboratories.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143999619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Production of p-anisaldehyde via whole-cell transformation using recombinant E. coli expressing trans-anethole oxygenase.","authors":"Zhikai Zhang, Qian Lin","doi":"10.2323/jgam.2025.02.001","DOIUrl":"10.2323/jgam.2025.02.001","url":null,"abstract":"<p><p>p-Anisaldehyde, a fragrance and flavour with important roles in food, cosmetics, and drug industries, is currently synthesized through chemical methods. Production of p-anisaldehyde by chemical oxidation of trans-anethole in industry gives rise to excessive by-products and adverse environmental impacts, whereas biological process would address such problems. Here, we presented a process of biotransformation of trans-anethole for production of p-anisaldehyde. The tao gene encoding for trans-anethole oxygenase (TAO) from Paraburkholderia sp. MR185 was fused with a solubilization tag GST and ProS2, respectively. GST did not exhibit solubility enhancement effect, whereas fusion with ProS2 significantly improved TAO's soluble expression in E. coli and the fusion protein ProS2-TAO-Sil3K accounted for more than 40% of total soluble proteins. ProS2-TAO-Sil3K was purified by simple silica affinity and its activity did not require addition of NADH, NADPH, and FAD. Metal ions Co<sup>2+</sup>, Zn<sup>2+</sup>, Ni<sup>2+</sup>, and Cu<sup>2+</sup> displayed significant inhibition effect on TAO activity, and addition of Fe<sup>2+</sup> improved enzyme activity by 32.6%. After induction, engineered E. coli cells were used as whole-cell biocatalyst for transformation of trans-anethole, and the final concentration of p-anisaldehyde reached 10.18 mM (1.38 g/L), with the volumetric productivity of 0.11 g/L/h and conversion rate of 67.9%. These results reveal that the biosynthesis of p-anisaldehyde has a great potential in practice.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Screening system for MAA-CoA productivity using 2-methylcitrate biosensor.","authors":"Satoshi Hasegawa, Ryota Kato, Daichi Ishihara, Miyu Tsukada, Takumi Ojima, Shigeko Kawai-Noma, Daisuke Umeno","doi":"10.2323/jgam.2025.05.003","DOIUrl":"10.2323/jgam.2025.05.003","url":null,"abstract":"<p><p>Methyl methacrylate (MMA), the primary raw material of acrylic resin, is an important polymeric material due to its increasing demand and ease of recycling. The most promising biosynthetic route for MMA involves the condensation of methanol with methacrylyl-CoA (MAA-CoA), an intermediate in the valine degradation pathway. The toxicity of MAA-CoA, poor stability and low activity of the heterologous pathway enzymes make this biosynthetic pathway less feasible. For enabling the evolutionary engineering of this pathway and its components (enzymes), we constructed a biosensor system in which the cellular level of key intermediate MAA-CoA can be evaluated in a high-throughput manner. With the aid of this MAA-CoA sensory system, we could establish the functional pathway from isobutyric acid to MAA-CoA. The sensor described in this paper should be valuable tool in the design-build-test-learn cycle for optimizing and breeding this MMA pathway.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144368912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}