Short-term dietary changes can result in mucosal and systemic immune depression.

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.

High level production of flavonoid rhamnosides by metagenome-derived Glycosyltransferase C in Escherichia coli utilizing dextrins of starch as a single carbon source.

Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides.

Activity-based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from grape.

Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl ß-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol ß-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity.

A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome.

Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol ß-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening.