| This dissertation selected Agrobacterium sp. strain, ATCC 31749 as a model system to explore an efficient fermentation strategy for the production ofβ-1,3-glucan (curdlan). There has been very limited genomic information on the Agrobacterium sp. ATCC 31749. The ETCs (Electron Transfer Chains) and metabolic network were constructed based on the comparative genomic method. The effects of DO (dissolved oxygen) gradients on the metabolic characteristics, gene expression profiles of Agrobacterium sp. were studied. Based on these results, an efficient curdlan production process (two-stage DO combined with pH control) was developed. In the second part of the Thesis (described in Chapter 5) a library of glucan oligosaccharides, obtained from various types of glucans after their partial depolymerization, was constructed. These oligosaccharides were converted into neoglycolipid (NGL) probes for generating glucan oligosaccharide microarrays. Microarray binding experiments were then carried out with two proteins the human immune system to determine the specificities of the interactions, including the minimum chain length and glucose linkage requirement for oligosaccharides.The main results were described as follows:1) With the PCR and gene sequence methodology, ten genes sequences of Agrobacterium sp. ATCC 31749 were acquired and analysis as well as the genomic specificity identification between Agrobacterium sp. ATCC 31749 and other 1000 bacterial genomes were undertaken. As the genome sequence of Agrobacterium sp. ATCC 31749 was not available when this work was undertaken, the primers were designed based on the highly conservative region of the same gene from different bacteria. The DNA fragments acquired by PCR were sequenced. On that basis, the sequences of eleven gene fragments from Agrobacterium sp. ATCC 31749 were determined. These sequences were highly consistent with that from previous reports. The eleven genes from Agrobacterium sp. ATCC 31749 and the genes from other four Agrobacterium which is sequenced successfully were compared on the basis of homology. The genome of Agrobacterium sp. ATCC 31749 is highly consistent with the genome of Agrobacterium tumefaciens C58 at 100% value. The respiration chain and metabolic network in Agrobacterium sp. ATCC 31749 was constructed base on these in Agrobacterium tumefaciens C58.2) To determine the mechanism of DO modulation of mRNA translation, the effect of DO gradients on the expression of genes related to the ETCs, TCA cycle and curdlan synthesis in Agrobacterium sp. ATCC 31749 were investigated under nitrogen limited condition using qRT-PCR. The transcriptional levels of the genes governing TCA cycle (icd, sdh B and mdh), curdlan synthesis (glm M and gal U) and ETC system (cyo A, cat D and fix N) increased concomitantly with the elevation of DO. Whereas, the transcriptional level of cytochrome d (cyd A) exposed to different DO was changed slightly. At 50% DO level, the mRNAs of cyo A, cat D, fix N, icd, sdh B, mdh, glm M and gal U were 3-6 times higher than those under other DO conditions studied. The results suggested that DO can apparently affect the translational levels of the genes particularly related to the metabolic network of glucose and ATP regeneration have similar modulation profile under 50% DO.3) Based on the fermentation technology with DO control, the effect of DO gradients combined with pH on curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan, and understanding of the mechanism of change of DO and the glucose metabolic network distribution to enhance the glucose metabolic flux for curdlan biosynthesis. The intracellular AMP, UMP, UDP-glucose, NADH and UTP at 60% DO is 1.4, 7.9, 4, 3, and 1.5-times, respectively, under 15% DO. The curdlan yield improved with the elevation of DO. The highest curdlan yield was 43 g/L under 60% DO which was 2.85 times higher than that of 5% DO condition (15 g/L).4) The DO concentration had apparent effect on curdlan production through enhancement of mRNA translational levels and intracellular nucleotide levels. Baseed on these results, a two-stage curdlan fermentation process has been developed and verified. The more optimal DO for curdlan production was between 45% and 60 %. The two-stage DO combine with pH (0-20 h, the pH was 7.0 after 20 h, the pH was controlled at 5.6; for DO levels, from 20 h to 50 h, 60% DO was suitable, thereafter 40% DO) was used. The resulting curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were improved by 28%, 30% and 20%, respectively.5) Series of gluco-oligosaccharide neoglycolipid (NGL) probes from 2mer to 13mer, includingβ-1,3,α-1,4,β-1,4,α-1,6 andβ-1,6-linkage, have been prepared in Prof. Ten Feizi’s laboratory. To complement currently available gluco-oligosaccharides and have a comprehensive series of the for microarray analysis,α-1,2-glucofructosides, cyclicβ-1,2-glucan,α-1,3-glucan and lentinan (β-1,3/β-1,6-branched glucan) were partially depolymerized by acid hydrolysis and fractionated by gel-filtration chromatography. Mass spectrometry and NMR were used for linkage and sequence analyses of the oligosaccharide fractions. Electrospray tandem mass spectrometry analysis of all the gluco-heptasaccharides revealed that different linkage-types produced different characteristic fragmentation. Howeverαorβanomeric configuration does not affect the fragmentation behaviour. Theαandβconfigurations can be readily assigned by 1H-NMR. In this way, all the oligosaccharide sequences were determined. NGL probes ( a total of 63 probes) were then synthesized from oligosaccharides obtained from different glucans before construction of glucan oligosaccharide microarrays. The microarrays were applied to analyze the linkage specificities and the minimum oligosaccharide chain lengths recognized by several recombinant proteins of the immune system, among them are Dectin-1 and DC-SIGN. As predicted Dectin-1 specifically bound toβ-1,3-gluco-oligosaccharides, and did not bind to any of theα-1,2-,α-1,3-,α-1,4-,α-1,6-,β-1,2-,β-1,4-,β-1,6-,β-1,3-β-1,4-gluco-oligosaccharides. The interactions of Dectin-1 with branched gluco-oligosaccharides, in particular those withβ-1,3/β1,6-linkages require further investigation. A particularly potent preparation of DC-SIGN was found to bind to gluco-oligosaccharides derived from bacteria, fungi or plants. The binding of DC-SIGN to gluco-oligosaccharides is a novel finding. Further investigation is ongoing in the Feizi laboratory to define the fine specificity, including the glucosyl linkage preference of DC-SIGN and the binding strength to gluco-oligosaccharides compared with other known DC-SIGN carbohydrate ligands. The results indicated that the position and frequency ofβ-1,6-branch play an important role for DC-SIGN interaction with gluco-oligosaccharides. |
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