ABSTRACT
Fluorescence-based methods are important tools for the analysis of nucleic acids in vitro and in cells. In this study, two cationic cyanine-styryl derivatives were produced using a two-step synthesis. Their optical properties were evaluated in different solvents, and frontier molecular orbital theory was utilized to interpret the findings. The DNA binding of these molecules was investigated to show fluorescence intensification. The molecular docking of both dyes in DNA illustrated the relevance of the electrostatic interaction between the quaternary ammonium of both dyes and the phosphate of the DNA backbone. Last but not least, applications of the synthesized styryl dyes were demonstrated to be selective towards cancer cells and particular kinds of bacteria.
ABSTRACT
ß-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 âµtktA âµtktB âµptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10â mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1â mM D-ribose supplied from the supernatant of E. coli MG1655 âµtktA âµtktB âµptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.
Subject(s)
Nicotinamide Mononucleotide , Xylose , Escherichia coli/genetics , Escherichia coli/metabolism , NAD/metabolism , Nicotinamide Mononucleotide/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Nucleotides/metabolism , Ribose , Xylose/metabolismABSTRACT
We designed and synthesized a fatty aldehyde surrogate containing a formyl thioester group, which can be reduced by fatty aldehyde reductase (FALR) with stoichiometric formaldehyde generation. It can be rapidly visualized and quantified using the Purpald assay. We demonstrated its successful application in the high throughput screening of FALR engineering.