| Carbohydrates constitute one of the major classes of bio-macromolecules in living organisms, they are not only essential components of cells but also the main energy source to maintain life activities. In addition, carbohydrates could exhibit irreplaceable roles as informative molecules in a variety of vital biological processes.So, Glycobiology which is an important science for understanding the life process and the development of human health, has been taken seriously by scientific community and biopharmaceutical companies.ABO blood group system is the most common blood type classification system,and can be divided into A, B, O and AB type. The antigens of ABO blood group which are glycoproteins and glycolipids essentially,can be decided by the compositions and structures of the terminal part of the carbohydrate chains. Now, the relationship of blood type and disease has become a research hotspot. Some reports showed that the incidences of some diseases are different in population with different blood groups, such as gastric cancer, pancreatic cancer, coronary heart disease,Hypertension, otitis media effusion and viral encephalitis. However, the related researches are still staying in statistics and analysis of cases, and the specific mechanism of interaction between blood group and diseases is not well understood.Because the blood group antigens are vital for researches on blood group and human health, the synthesis of these carbohydrate antigens and other glycoconjugates with biological activities is particularly important.Because enzymatic synthesis is considered more attractive with the advantages of strone regio- and stereo-specificity, economic and time saving, synthesis of structure-defined glycans by glycosyltransferases has become an attractive alternative to the chemical synthesis. However, the limited quantities and expensive price of sugar nucleotides which are the substrates of glycosyltransferases restrict the application of enzymatic synthesis in preparing bioactive oligosaccharides and thus hinder the research and application of glycoconjugates in the field of biological medicine.Sugar nucleotides, known as active sugars, are constructed by linking the anomeric carbon of monosaccharides to nucleoside diphosphates or nucleoside monophosphates. They can act as sugar donors to explore the biochemical characterization of glycotransferases. Besides, analogs of natural sugar nucleotides may be inhibitor to enzymes, or can be used for analyzing the glycoconjugate biosynthetic pathways. Enzymatic synthesis and microbiological fermentation have their own advantages and have the potential to achieve the industrial production of sugar nucleotides.Gut microbiota which is an indivisible component of human body, comes into play like an important functional organs and has effect on metabolism,immunoregulation, defense, brain development and human behavior.Intestinal dysbacteriosis is associated with the occurrence and development of various diseases. In the coevolution process of gut microbiota and the host, the composition and structure of gut microbiota would be affected by a variety of factors from host genotype,diet, environmental exposures,social behavior and so on. Bacterial surfaces are coated with complex polysaccharides, including lipopolysaccharides (LPSs),capsular polysaccharides (CPSs) and extracellular polysaccharide (EPSs). Many of them was reported to exhibit specific blood group A, B, or H(O) activity and possess a close serological relationship to A, B, and H(O) erythrocyte agglutinogens. Therefore,the explorations to the relationship of ABO blood group and gut microbiota could not only explain the correlation of blood type and disease susceptibility, but also provide a convincing evidence for the hypothesis of the origin of ABO natural antibodies.One aim of this thesis is to simplify the enzymatic method of UDP-GlcNAc synthesis,and obtain the single product through a small scale preparation. The availability of UDP-GlcNAc is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK.After determination of the optimum catalytic condition (pH 8.0 at 40 C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 %from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.GDP-Fuc is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-Fuc biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides,the biosynthesis of GTP,an essential substrate for GDP-Fuc biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-Fuc content of 4.6±0.22 ?mol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-Fuc content further rose to 6.6±0.14 ?mol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-Fuc biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-Fuc analogues encompassing chemical modification on the fucose residue.The factors which influence the gut microbiota composition are diverse. Because of the specific blood group A, B, or H(O) activity of some bacteria polysaccharides,the impact of blood group on gut microbiota composition has our full attention. In chapter 4, we analyzed 16S rDNA gene sequences from 171 individuals with different ABO blood groups which have the same body mass index, age and gender. Although the principal compositions of gut species are similar among different blood groups,there are some differences at the different division levels. The differences mainly exist in 3 phyla, phylum bacteroidetes, phylum synergistetes and phylum tenericutes.In the other phyla, there is no significant difference at the phylum level, however, the abundance of some genera shows difference. We also observed a significant decrease in a diversity in blood group A individual's gut microbiome, suggesting that the microbial diversity in blood group A is lower than that in others. In addition, we found the amount of probiotics in blood group A and B individuals was lower than that in other blood type individuals. In order to further understand how the intestinal bacteria adapt to different selective pressure from the host as well as from microbial competitors, we detected the blood group A and B antigens activity of the intestinal bacteria. Intestinal bacteria in blood group A present more blood group A antigen activity, intestinal bacteria in blood group B exist more blood group B antigen activity,intestinal bacteria in blood group O and AB exist equal activity of blood group A and B antigen, suggesting that the ABO blood groups antigen may affect the composition of gut microbiome and thus further influence the interaction of intestinal microbiome and host. The result may indicate that ABO blood group is one of the host factors modulating the composition of the human intestinal microbiota and may provide a new explication for the relationship between ABO blood groups and diseases, thus enabling new applications in the field of personalized nutrition and medicine.In summary,in this thesis two kinds of sugar nucleotides were synthesized via enzymatic synthesis and microbial fermentation. We specifically linked ABO blood group to the composition of gut microbiota and offered a preliminary explanation to the mutual relation between them in terms of the similarity of some bacterial polysaccharides and blood group antigens. |
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