Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
This paper focuses on the biological and chemical variability of four yacon (Smallanthus sonchifolius) accessions cultivated under field conditions. Significant variations in tuber shape, weight, content of oligofructans, as well as in leaf isozymes, phenolics, and relative DNA contents were found. Accessions 6 and 88 were the most productive (up to 3.01 and 3.74 kg/plant); accession 48 was the most balanced from the yield aspect in three vegetative periods. A significantly higher content of beta-(2-->1) oligofructans was noted in accessions 48 and 88 as compared to 6 and 60. No difference in sucrose, glucose, and fructose level was observed. Only accession 6 exhibited separate acid phosphatase and esterase isoforms. Accessions 6 and 60 had the highest content of phenolics, and accession 88 had the lowest relative DNA content. Large yacon intraspecific variation may be useful in future detailed research as a good background for breeding, growing, and utilization in industrial processing.
HighlightsMethod for simultaneous determination of individual apple procyanidins and catechins is presented.Procyanidins separated on a HILIC column.Accurate quantification achieved using isolated procyanidin oligomers.Method validated via an inter-laboratory evaluation.
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