Overexpression of endogenous 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in cyanobacterium Synechocystis sp. PCC6803 accelerates protein aggregation.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for the production of precursors of all isoprenoids. In a previous study, we had examined the overexpression of an endogenous DXS in a Synechocystis sp. PCC6803 mutant (DXS_ox), and found that the dxs mRNA level was 4-fold higher than that in the wild-type (WT) strain. However, the DXS protein level was only 1.5-fold higher, leading to the assumption that the level might be regulated by post-transcriptional events. In this study, we have additionally introduced an exogenous isoprene synthase (IspS; which can release MEP pathway products from the cell as gaseous isoprene) into the WT and DXS_ox strains (WT-isP and DXSox-isP strains, respectively), and their detailed DXS expression profiles were investigated from the induction phase through to the late-logarithmic phase. In the induction phase, the isoprene productivity of the DXSox-isP strain was slightly but significantly (1.4- to 1.8-fold) higher than that of the WT-isP strain, whereas the levels were comparable in the other phases. Interestingly, the ratios of soluble:insoluble DXS protein were remarkably low in the DXSox-isP strain during the induction phase to the early-logarithmic phase, resulting in a moderate level of soluble DXS. All our results suggested that the high translation rate of DXS disturbs the refolding process of DXS. To enhance the concentration of the active DXS in cyanobacteria, the enhancement of the DXS maturation system or the introduction of exogenous and robust DXS proteins might be necessary.

Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

BACKGROUND: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. METHODS: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). RESULTS: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. CONCLUSIONS: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.

Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.