Awakening the natural capability of psicose production in Escherichia coli.

Due to the rampant rise in obesity and diabetes, consumers are desperately seeking for ways to reduce their sugar intake, but to date there are no options that are both accessible and without sacrifice of palatability. One of the most promising new ingredients in the food system as a non-nutritive sugar substitute with near perfect palatability is D-psicose. D-psicose is currently produced using an in vitro enzymatic isomerization of D-fructose, resulting in low yield and purity, and therefore requiring substantial downstream processing to obtain a high purity product. This has made adoption of D-psicose into products limited and results in significantly higher per unit costs, reducing accessibility to those most in need. Here, we found that Escherichia coli natively possesses a thermodynamically favorable pathway to produce D-psicose from D-glucose through a series of phosphorylation-epimerization-dephosphorylation steps. To increase carbon flux towards D-psicose production, we introduced a series of genetic modifications to pathway enzymes, central carbon metabolism, and competing metabolic pathways. In an attempt to maximize both cellular viability and D-psicose production, we implemented methods for the dynamic regulation of key genes including clustered regularly interspaced short palindromic repeats inhibition (CRISPRi) and stationary-phase promoters. The engineered strains achieved complete consumption of D-glucose and production of D-psicose, at a titer of 15.3 g L-1, productivity of 2 g L-1 h-1, and yield of 62% under test tube conditions. These results demonstrate the viability of whole-cell catalysis as a sustainable alternative to in vitro enzymatic synthesis for the accessible production of D-psicose.

Alternative Identification of Glycosides Using MS/MS Matching with an In Silico-Modified Aglycone Mass Spectra Library.

Glycosylation of metabolites serves multiple purposes. Adding sugars makes metabolites more water soluble and improves their biodistribution, stability, and detoxification. In plants, the increase in melting points enables storing otherwise volatile compounds that are released by hydrolysis when needed. Classically, glycosylated metabolites were identified by mass spectrometry (MS/MS) using [M-sugar] neutral losses. Herein, we studied 71 pairs of glycosides with their respective aglycones, including hexose, pentose, and glucuronide moieties. Using liquid chromatography (LC) coupled to electrospray ionization high-resolution mass spectrometry, we detected the classic [M-sugar] product ions for only 68% of glycosides. Instead, we found that most aglycone MS/MS product ions were conserved in the MS/MS spectra of their corresponding glycosides, even when no [M-sugar] neutral losses were observed. We added pentose and hexose units to the precursor masses of an MS/MS library of 3057 aglycones to enable rapid identification of glycosylated natural products with standard MS/MS search algorithms. When searching unknown compounds in untargeted LC-MS/MS metabolomics data of chocolate and tea, we structurally annotated 108 novel glycosides in standard MS-DIAL data processing. We uploaded this new in silico-glycosylated product MS/MS library to GitHub to enable users to detect natural product glycosides without authentic chemical standards.

Synthesis of Spirobicyclic Pyrazoles by Intramolecular Dipolar Cycloadditions/[1s, 5s] Sigmatropic Rearrangements.

The formation of fused pyrazoles via intramolecular 1,3-dipolar cycloadditions of diazo intermediates with pendant alkynes is described. A subsequent thermal [1s, 5s] sigmatropic shift of these pyrazole systems resulted in a ring contraction, forming spirocyclic pyrazoles. The limitations of this rearrangement were explored by changing the substituents on the nonmigrating aromatic ring and by using substrates lacking an aromatic linkage to the propargyl group.