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.

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.

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-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 Co²⁺, Zn²⁺, Ni²⁺, and Cu²⁺ displayed significant inhibition effect on TAO activity, and addition of Fe²⁺ 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.

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.

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 NifD₂K₂, 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 NifD₂K₂ component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD₂K₂ formation, effectively increased nitrogenase activity.

Zn-deficiency, a global health challenge affects one-third of the world population. Zn-biofertilizer offer an efficient and cost-effective remedy. As Zn-biofertilizer can improve plant growth and grain’s Zn-content ensuring improved dietary Zn-supply. This study sought to understand how silver and TiO₂ nanoparticles in the rhizosphere affect the activity of Zn-solubilization bacteria (ZSB) and plant growth. Two ZSB strains Bacillus sp. D-7 and Pseudomonas sp. D-117 with excellent Zn-solubilization efficiency of 254 and 260%, respectively were isolated and characterized using polyphasic characterization including 16S rRNA gene sequencing to formulate an effective Zn-biofertilizer. The plant growth promoting activity of this biofertilizer in Mung bean was checked in the presence and absence of various doses of TiO₂ and Ag-NPs and was compared with plant grown without biofertilizer. The change in rate of seed germination, vegetative growth (shoot and root length, fresh and dry weight), photosynthetic pigment and Zn-content was checked. Lower doses of nanomaterials (50 and 100 mg kg⁻¹ soil) slightly promoted the plant growth compared to control. While, higher doses (200 and 400 mg kg⁻¹ soil) inhibited the growth. A maximum decrease of shoot length, root length, fresh-weight, and dry-weight of 57.1, 53.9, 53.1, and 10.4% respectively was observed with 400 mg kg⁻¹ of Ag-NPs. However, in the presence of ZSB, the decrease at the same Ag-NP concentration was 41.6, 31.5, 27.4, and 6.6, respectively. These results strongly suggest that Zn-solubilizing bacteria improve resilience to nanoparticles toxicity and helps in Zn fortification in Mung bean even under nanomaterial stress.