●Inositol

Inositol stands for a group of cyclohexanehexol consisting of 9 stereoisomers epimerizing the six hydroxyl groups. Among them, myo-inositol is most abundant in nature and can be supplied at relatively low prices as produced from cheap materials such as rice bran. The other inositol stereoisomers are rare and expensive, but some are known to exert specific physiological effects beneficial for human health. For example, D-chiro-inositol and pinitol (3-O-methyl-D-chiro-inositol) have an insulin-like effect to lower the blood sugar level and are also useful to cure polycystic ovary syndrome. On the other hand, scyllo-inositol has an effect to suppress the aggregation of β-amyloid in the brain, and thus is expected as a remedy for Alzheimer's disease.

●Inositol metabolism in B. subtilis

・Metabolic engineering of B. subtilis for the bio-production of rare inositol
・Understanding the mechanism of NADPH regeneration in B. subtilis
・Functional modification of inositol dehydrogenase
・Elucidation of enzymological and physiological significance of functionally unknown iolH gene

We have established metabolic engineering of B. subtilis to convert myo-inositol into scyllo-inositol, a promising therapeutic agent for Alzheimer's disease. In addition, to better understand the deeper cellular system required for the inositol conversion, we are elucidating the NADPH regeneration necessary as a coenzyme for the key enzyme IolW
An improved B. subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer’s disease

●Secretion of useful proteins by B. subtilis

・B. subtilis cells displaying the artificially-designed cellulosome on the surface.

Cellulose and hemicellulose are main constituents of non-edible plant biomass and hardly decomposable materials. We aim to create a cellulose-degrading B. subtilis cells displaying the artificially designed cellulosome on the surface.

・Improvement of B. subtilis phytase secretion

Phytic acid is rich in rice bran as the major storage form of phosphates of cereals and legumes. We aim at improving the efficiency of secretory production of phytase, which produces myo-inositol by trimming off phosphate from phytic acid in rice bran.

●Inositol metabolism in thermophilic Geobacillus kaustophilus HTA426

・Elucidation of physiological significance of inositol metabolism in G. kaustophilus HTA426
・Regulation of the genes involved in inositol degradation in G. kaustophilus HTA426

G. kaustophilus can grow using myo-inositol as the sole carbon source. However, the genes and their regulation involved in the growth mechanism have not been fully elucidated. We are pursuing the possibility to utilize G. kaustophilus for the bio-production of rare inositol.
Three inositol dehydrogenases involved in the consumptionn and interconversion of inositol stereoisomers in a thermophilic bacterium, Geobacillus kaustophilus HTA426

●Development of efficient transformation method of thermophilic bacteria G. kaustophilus

G. kaustophilus is an industrially beneficial bacterium. Nevertheless, conventional transformation methods are inefficient and time consuming for this bacterium. We developed a novel method for transforming G. kaustophilus using conjugation plasmid pLS20cat.
A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus

●Application of conjugative genetic transfer among Gram-positive bacteria

The conjugative plasmid, pLS20, isolated from B. subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. The oriT sequence of pLS20cat (oriTLS20) was eliminated to obtain the plasmid, pLS20catΔoriT. pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriTLS20, after mixing the liquid cultures of the donor and recipient for only 15 min. This will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.
Rapid conjugative mobilization of a 100 kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for self-transfer

●Bacillus velzensis S141: Promoting symbiotic nitrogen fixing capacity of soybean root nodule bacteria

Soybean symbiont Bradyrhizobium diazoefficience is known to form root nodules for a symbiotic nitrogen fixation with soybean. In a previous study, B. velzensis S141 was isolated as a plant growth-promoting rhizobacterium which enhances the beneficial effect of B. diazoefficience on soybean growth. We aim at revealing the underlying mechanism for the enhancement of the symbiotic nitrogen fixation of B. diazoefficience by B. velzensis S141, in collaboration with Suranaree University of Technology, Thailand.

●PHB production by B. diazoefficience

Soybean symbiont B. diazoefficience accumulates a large amount of poly-β-hydroxybutyrate (PHB) in cells, which is one of the promising bioplastics. PHB biosynthesis requires several enzymes involved in the synthesis and the decomposition of PHB. In addition, we found that other proteins called phasins, which stabilize the growing PHB granules, might play an important role in the PHB accumulation. We aim at elucidating the mechanism for the efficient PHB accumulation in B. diazoefficience.
PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum
Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation

●Transformation of Leuconostoc genus

Leuconostoc are obligatory heterofermentive lactic acid bacteria. In spite of its industrial importance, at present genetic manipulation of Leuconostoc is difficult because of its extremely low transformation efficiency. We aim at establishing its versatile transformation (mainly by protoplast method) using Leu. mesenteroides as a model organism.