Synthesis of two derivatives of tigogenyl disaccharides containing N-acetylglucosamine and the long range shielding effect of benzoyl groups

Two tigogenyl glycosides containing N-acetylglucosamine were synthesized. Their structures were confirmed by ^1H and ^13C NMR spectra. The shielding effect caused by benzoyl groups was elucidated by ^1H NMR, COSY, HSQC, HMBC spectroscopy.

Production of N-acetylglucosamine with Vibrio alginolyticus FA2,an emerging platform for economical unsterile open fermentation

Members of the Vibrionaceae family are predominantly fast-growing and halophilic microorganisms that have captured the attention of researchers owing to their potential applications in rapid biotechnology.Among them,Vibrio alginolyticus FA2 is a particularly noteworthy halophilic bacterium that exhibits superior growth capability.It has the potential to serve as a biotechnological platform for sustainable and eco-friendly open fermentation with seawater.To evaluate this hypothesis,we integrated the N-acetylglucosamine(GlcNAc)pathway into V.alginolyticus FA2.Seven nag genes were knocked out to obstruct the utilization of GlcNAc,and then 16 exogenous gna1s co-expressing with EcglmS were introduced to strengthen the flux of GlcNAc pathway,respectively.To further enhance GlcNAc production,we fine-tuned promoter strength of the two genes and inactivated two genes alsS and alsD to prevent the production of acetoin.Furthermore,unsterile open fermentation was carried out using simulated seawater and a chemically defined medium,resulting in the production of 9.2 g/L GlcNAc in 14 h.This is the first report for de-novo synthesizing GlcNAc with a Vibrio strain,facilitated by an unsterile open fermentation process employing seawater as a substitute for fresh water.This development establishes a basis for production of diverse valuable chemicals using Vibrio strains and provides insights into biomanufacture.

Synergistic utilization of carbon sources for efficient biosynthesis of N-acetylglucosamine

N-acetylglucosamine(GlcNAc)is an amino monosaccharide that has a variety of bioactivities and is widely used in pharmaceutical and food industries.Production of GlcNAc by chitin hydrolysis is limited by the supply of raw materials and encounters the risk of shellfish protein contamination.For efficient biosynthesis of GlcNAc,one challenge is to balance the carbon distribution between growth and production.Here,we applied the strategy of synergistic carbon utilization,in which glycerol supports cell growth and provides the acetyl group of GlcNAc while glucose serves as the precursor to glucosamine.The efficiency of GlcNAc production was stepwise improved by blocking the product re-uptake and degradation,strengthening the biosynthetic pathway and synergistically utilizing two carbon sources.With these efforts,the final strain produced 41.5 g/L GlcNAc with a yield of 0.49 g/g of total carbon sources.In addition,we also explored the feasibility of using acetate as a cheap carbon source to partly replace glycerol.This study provides a promising alternative strategy for sustainable and efficient pro-duction of GlcNAc.

Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis

Glucosamine(GlcN)and its acetylated derivative N-acetylglucosamine(GlcNAc)are widely used in the pharmaceutical industries.Here,we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum.Specifically,we ligated the GNA1 gene,which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate,into the plasmid pJYW4 and then transformed this recombinant vector into the C.glutamicum ATCC 13032,ATCC 13869,ATCC 14067,and S9114 strains,and we assessed the GlcNAc titers at 0.5 g/L,1.2 g/L,0.8 g/L,and 3.1 g/L from each strain,respectively.This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C.glutamicum strains.Therefore,we performed whole genome sequencing of the S9114 strain,which has not been previously published,and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested.Next,nagA(encoding GlcNAc-6-phosphate deacetylase)and gamA(encoding GlcN-6-phosphate deaminase)were deleted in C.glutamicum S9114 to block the catabolism of intracellular GlcNAc,leading to a 54.8%increase in GlcNAc production(from 3.1 to 4.8 g/L)when grown in a shaker flask.In addition,lactate synthesis was blocked by knockout of ldh(encoding lactate dehydrogenase);thus,further increasing the GlcNAc titer to 5.4 g/L.Finally,we added a key gene of the GlcN synthetic pathway,glmS,from different sources into the expression vector pJYW-4-ceN,and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L.This is the first report concerning the metabolic engineering of C.glutamicum,and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.

Synthesis of an Ursolic Acid Saponin with N-Acetylglucosamine-containing Trisaccharide Residue

The focus of this work is the synthesis of an ursolic acid saponin with an N-acetylglucosamine-containing trisaccharide residue. Therefore, ursolic acid 3-yl α-L-arabinopyranosyl-（1→2）-α-L-arabinopyranosyl-（1→6）-2- acetamido-2-deoxy-β-D-glucopyranoside （1） was concisely synthesized in convergent synthesis with 48.0% overall yield. The structure of saponin 1 was confirmed by ^1H NMR, ^13C NMR and mass spectra.

Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry

Bisecting N-acetylglucosamine(GlcNAc),a GlcNAc linked to the coreβ-mannose residue via aβ1,4 linkage,is a special type of N-glycosylation that has been reported to be involved in various biological processes,such as cell adhesion and fetal development.This N-glycan structure is abundant in human trophoblasts,which is postulated to be resistant to natural killer cellmediated cytotoxicity,enabling a mother to nourish a fetus without rejection.In this study,we hypothesized that the human amniotic membrane,which serves as the last barrier for the fetus,may also express bisected-type glycans.To test this hypothesis,glycomic analysis of the human amniotic membrane was performed,and bisected N-glycans were detected.Furthermore,our proteomic data,which have been previously employed to explore human missing proteins,were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed.A total of 41glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc,and 25 of these glycoproteins were reported to exhibit this type of modification for the first time.These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane,and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.

Synthesis of 4-[F-18]Fluoro-4-Deoxy-N-Acetyl-1,3,6-Tri-O-Acetylglucosamine, a Potential Brain Imaging PET Agent

The synthesis of a promising brain imaging agent 4-[F-18]fluoro-4-deoxy-N-acetyl-1,3,6-tri-O-acetylglucosamine, 2, was successfully accomplished from commercially available N-acetyl glucosamine in 5 steps. The non-decay corrected radiochemical yield and purity were found to be 31% ± 4% (n = 3) and >98% respectively. The total reaction time for radio labelling step was 50 min.

Systematic analyses of glutamine and glutamate metabolisms across different cancer types

Background: Glutamine and glutamate are known to play important roles in cancer biology. However, no detailed information is available in terms of their levels of involvement in various biological processes across different cancer types, whereas such knowledge could be critical for understanding the distinct characteristics of different cancer types. Our computational study aimed to examine the functional roles of glutamine and glutamate across different cancer types.Methods: We conducted a comparative analysis of gene expression data of cancer tissues versus normal control tissues of 11 cancer types to understand glutamine and glutamate metabolisms in cancer. Specifically, we developed a linear regression model to assess differential contributions by glutamine and/or glutamate to each of seven biological processes in cancer versus control tissues.Results: While our computational predictions were consistent with some of the previous observations, multiple novel predictions were made:(1) glutamine is generally not involved in purine synthesis in cancer except for breast cancer, and is similarly not involved in pyridine synthesis except for kidney cancer;(2) glutamine is generally not involved in ATP production in cancer;(3) glutamine's contribution to nucleotide synthesis is minimal if any in cancer;(4) glutamine is not involved in asparagine synthesis in cancer except for bladder and lung cancers; and(5) glutamate does not contribute to serine synthesis except for bladder cancer.Conclusions: We comprehensively predicted the roles of glutamine and glutamate metabolisms in selected metabolic pathways in cancer tissues versus control tissues, which may lead to novel approaches to therapeutic development targeted at glutamine and/or glutamate metabolism. However, our predictions need further functional validation.