Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Two cyanobacterial response regulators with diguanylate cyclase activity, Rre2 and Rre8, participate in biofilm formation.

Phototrophic bacteria face diurnal variations of environmental conditions such as light and osmolarity that affect their carbon metabolism and ability to generate organic compounds. The model cyanobacterium, Synechocystis sp. PCC 6803 forms a biofilm when it encounters extreme conditions like high salt stress, but the molecular mechanisms involved in perception of environmental changes that lead to biofilm formation are unknown. Here, we studied two two-component regulatory systems (TCSs) that contain diguanylate cyclases (DGCs), which produce the second messenger c-di-GMP, as potential components of the biofilm-inducing signaling pathway in Synechocystis. Analysis of single mutants provided evidence for involvement of the response regulators, Rre2 and Rre8 in biofilm formation. A bacterial two-hybrid assay showed that Rre2 and Rre8 each formed a TCS with a specific histidine kinase, Hik12 and Hik14, respectively. The in vitro assay showed that Rre2 had DGC activity regardless of its de/phosphorylation status, whereas Rre8 required phosphorylation for DGC activity. Hik14-Rre8 likely functioned as an inducible sensing system in response to environmental change. Biofilm assays with Synechocystis mutants suggested that pairs of hik12-rre2 and hik14-rre8 responded to high salinity-induced biofilm formation. Inactivation of hik12-rre2 and hik14-rre8 did not affect the performance of the light reactions of photosynthesis. These data suggest that Hik12-Rre2 and Hik14-Rre8 participate in biofilm formation in Synechocystis by regulating c-di-GMP production via the DGC activity of Rre2 and Rre8.

Associations between physical physique/fitness in children and bone development during puberty: a 4-year longitudinal study.

Bone growth is most remarkable during puberty. This study aimed to clarify the effects of physique and physical strength on bone mineral density and bone metabolism markers during puberty to help improve bone growth during puberty and prevent future osteoporosis. There were 277 pubertal participants (125 boys and 152 girls) in this survey from 2009 to 2015, all aged 10/11 and 14/15 years. The measures included physical fitness/physique indices (such as muscle ratio etc.), grip strength, bone density (osteo sono-assessment index, OSI), and bone metabolism markers (bone-type alkaline phosphatase and type I collagen cross-linked N-telopeptide). At 10/11-years-old for girls, a positive correlation was found between body size/grip strength and OSI. At 14/15-year-old for boys, all body size factors/grip strength were positively correlated with OSI. The change in body muscle ratio was positively correlated with change in OSI for both sexes. The height, body muscle ratio and grip strength at 10/11-year-old were significantly associated with OSI (positively) and bone metabolism markers (negatively) at 14/15-year-old for both sexes. Adequate physique building after 10/11 years for boys and before 10/11 years for girls may be effective in increasing peak bone mass.