Expression of clMagR/clCry4 protein in mBMSCs provides T2-contrast enhancement of MRI.

Here, we propose for the first time the evaluation of magnetosensitive clMagR/clCry4 as a magnetic resonance imaging (MRI) reporter gene that imparts sensitivity to endogenous contrast in eukaryotic organisms. Using a lentiviral vector, we introduced clMagR/clCry4 into C57BL/6 mice-derived bone marrow mesenchymal stem cells (mBMSCs), which could specifically bind with iron, significantly affected MRI transverse relaxation, and generated readily detectable contrast without adverse effects in vivo. Specifically, clMagR/clCry4 makes mBMSCs beneficial for enhancing the sensitivity of MRI-R2 for iron-bearing granules, in which cells recruit exogenous iron and convert these stores into an MRI-detectable contrast; this is not achievable with control cells. Additionally, Prussian blue staining was performed together with ultrathin cell slices to provide direct evidence of natural iron-bearing granules being detectable on MRI. Hence, it was inferred that the sensitivity of MRI detection should be correlated with clMagR/clCry4 and exogenous iron. Taken together, the clMagR/clCry4 has great potential as an MRI reporter gene. STATEMENT OF SIGNIFICANCE: In this study, we propose the evaluation of magnetosensitive clMagR/clCry4 as an MRI reporter gene, imparting detection sensitivity to eukaryotic mBMSCs for endogenous contrast. At this point, the clMagR and clCry4 were located within the cytoplasm and possibly influence each other. The clMagR/clCry4 makes mBMSCs beneficial for enhancing the sensitivity of MRI-R2 for iron-bearing granules, in which protein could specifically bind with iron and convert these stores into MRI-detectable contrast; this is not achieved by control cells. The viewpoint was speculated that the clMagR/clCry4 and exogenous iron were complementary to each other. Additionally, Prussian blue staining was performed together with TEM observations to provide direct evidence that the iron-bearing granules were sensitive to MRI.

Transcriptomic profiling reveals MEP pathway contributing to ginsenoside biosynthesis in Panax ginseng.

BACKGROUND: Panax ginseng C. A. Mey is one of famous medicinal herb plant species. Its major bioactive compounds are various ginsenosides in roots and rhizomes. It is commonly accepted that ginsenosides are synthesized from terpene precursors, IPP and DMAPP, through the cytoplasmic mevalonate (MVA) pathway. Another plastic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was proved also contributing to ginsenoside generation in the roots of P. ginseng by using specific chemical inhibitors recently. But their gene expression characteristics are still under reveal in P. ginseng. With the development of the high-throughput next generation sequencing (NGS) technologies, we have opportunities to discover more about the complex ginsenoside biosynthesis pathways in P. ginseng. RESULTS: We carried out deep RNA sequencing and comprehensive analyses on the ginseng root samples of 1-5 years old and five different tissues of 5 years old ginseng plants. The de novo assembly totally generated 48,165 unigenes, including 380 genes related to ginsenoside biosynthesis and all the genes encoding the enzymes of the MEP pathway and the MVA pathway. We further illustrated the gene expression profiles related to ginsenoside biosynthesis among 1-5 year-old roots and different tissues of 5 year-old ginseng plants. Particularly for the first time, we revealed that the gene transcript abundances of the MEP pathway were similar to those of the MVA pathway in ginseng roots but higher in ginseng leaves. The IspD was predicated to be the rate-limiting enzyme in the MEP pathway through both co-expression network and gene expression profile analyses. CONCLUSIONS: At the transcriptional level, the MEP pathway has similar contribution to ginsenoside biosynthesis in ginseng roots, but much higher in ginseng leaves, compared with the MVA pathway. The IspD might be the key enzyme for ginsenoside generation through the MEP pathway. These results provide new information for further synthetic biology study on ginsenoside metabolic regulation.

Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids.

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the ß-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH-cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.