Biosynthesis Of Chitooligosaccharides And Its Derivatives By Recombinant E. Coli Strains

Oligosaccharides are found in nature as components of a broad range of molecular structures, such as cell surface glycoproteins and glycolipids. Carbohydrate moieties of these structures play vital roles in cellular communication processes, as points of attachment for antibodies and other proteins, and as receptors for bacteria and viral particles. Oligosaccharides and glycoconjiugates therefore have enormous potential as therapeutic agents. However, this potential is far from being realized due to the complex structure of oligosaccharides which makes classical chemical synthesis difficult. Chemical synthesis of oligosaccharides has been enormously improved in the past 20 years making increasingly large structures available for biological studies. This still involves a large number of steps including many protections and deprotections despite some recent promising improvements such as solid supported synthesis. Interesting alternatives to fully chemical syntheses are chemo-enzymatic approaches, where the selectivity of enzymatic reactions is used to produce complex oligosaccharide structures. Enzymatic synthesis using glycosyltransferases of the Leloir pathway could circumvent the drawbacks of the chemical methods. Glycosyltransferases involved in the biosynthesis of such oligosaccharide structures showed highly stereo- and regiospecific bond formation, and almost no side products were formed during the reactions without protections.In response to the release by host plants of appropriate inducers, rhizobia synthesize and secrete a family of lipochitooligosaccharides (LCOs) called Nod factors. The first step in Nod factor assembly is performed by an N-acetylglucosaminyltransferase encoded by nodC. Chain elongation by NodC takes place at the nonreducing terminus. Then the deacetylase NodB removes the N-acetyl moiety from the nonreducing terminus of the N-acetylglucosamine oligosaccharides. Finally, an acyltransferase encoded by nodA links the acyl chain to the acetyl-free C-2 carbon of the nonreducing end of the oligosaccharide. The synthesis of chitin oligosaccharides by NodC proceeds by the addition of GlcNAc residues from the donor UDP-GlcNAc to O-4 of the nonreducing-terminal residue of the growing chain.Metabolic engineering of microbial cells represents one of the most promising strategies for deriving oligosaccharides in sufficient quantities to support research and clinical development. The synthesis process based on these microbes is scalable as it avoids expensive starting materials. An alternative way to produce oligosaccharides is in vivo production in recombinant Escherichia coli expressing glycosyltransferases. In this fermentation procedure, oligosaccharides could be produced extracellularly or intracellularly during cell growth using the machinery for sugar nucleotide synthesis in the cells. One important advantage of the whole-cell approach is the scalability. Since it avoids expensive starting materials and enzyme isolation, once the strain is developed, it can be easily scaled-up in a fermentor to produce the needed quantity of compounds.The key problem to be solved is to increase the productivity of oligosaccharides. A series of factors related to its productivity synthesized in vivo would be considered, such as activities of recombinant glycosyltransferase, cell density in cultures, pool levels of nucleotide sugar and oligosaccharides. It is very important to evaluate its actions in biosynthesis of oligosaccharides in vivo. Bacterial glycosyltransferases showed broad acceptor specificity. Therefore, bacterial enzymes were widely used for the synthesis of carbohydrate derivatives.It has already shown that Escherichia coli cells harboring recombinant NodC (. N-acetylglucosaminyltransferase) accumulated high intracellular concentrations of tetra-N-acetyl-chitopentase and penta-N-acetyl-chitopentaose. Cultivation of E. coli harboring heterologous gene nodC from A. caulinodans, Samain et al obtained a high yield of COs production, the COs accumulation can account for up to 5% of the total cell dry weight. However, the high production had to be performed at low growth rate with consequently a low UDP-GlcNAc requirement for cell biosynthesis, which lead to a low productivity.The fact that allyl 2-acetamido-2-deoxy-β-D-glucopyranoside as starting sugar can be used with recombinant Escherichia coli strains expressing nodC gene in vitro has already been described. We herein described the in vivo biosynthesis of chitin oligosaccharides and its derivatives by a batch culture of E. coli strain DH5α containing a plasmid carrying the cloned nodC gene.Construction of an in vivo biosynthesis system To evaluate the yield of COs synthesis in recombinant E. coli cells, a series of expression vector of NodC were constructed: NodC from M. loti: pUC-CL3, pSK-CL2, pET-CL1. NodC from S. meliloti: pUC-CM3. NodC from A.caulinodans: pC5 (pUC-CA, kindly provided by E. Samain). Cultivation results in flask showed that DH5a was the best E. coli strain for biosynthesis of COs. Comparison of the cultivation results in different recombinant E. coli strains harboring NodCL expression vector showed that DCL3 harboring pUC-CL3 has the highest yield, indicating that effective biosynthesis of COs in vivo required low expression of NodC. COs production is cell growth associated, biosynthesis of COs is related to the intracellular UDP-GlcNAc pool level. In bacteria, the donor, UDP-GlcNAc, is also an important intermediate for cell wall biosynthesis, extra expression levels of NodC would be increase cell burden.Effect of carbon source on UDP-GlcNAc level and COs yield. To improve the production of COs, the intracellular UDP-GlcNAc level was investigated (Fig 1). The results indicated that the intracellular UDP-GlcNAc has direct relation with the COs production in recombinant E.coli. Adding yeast extract may improve the intracellular UDP-GlcNAc level, which resulting an increased COs production. Substituting glycerol with glucose in MMYG medium, the COs yield was obviously decreased, but cell growth rate was increased. However, the intracellular UDP-GlcNAc level at this condition was low, indicating that the cell growth also consumed UDP-GlcNAc. Adding the direct precursor GlcNAc in the medium may increase both UDP-GlcNAc level and COs production.Biosynthesis of COs with One-Step feeding cultivation by E. coli strain of DCL3. During the one-step cultivation, the production of COs is cell growth associated . With the growth of recombinant E.coli, the COs accumulated to 775 mg.L^-1 in 30 h cultivation; the final cell concentration is 16.6 g.L^-1. The intracellular COs content kept at about 46-47 mg.g^-1 cell dry weight during the all fermentation process. Biosynthesis of COs with Two-Step feeding cultivation by E. coli strain ofDCL3. To increase the COs productivity, a two-step feed batch fermentation was employed. During the initial step, glucose was fed. Cells are growing very fast and the cell dry weight reached 16 g.L^-1 within 14 hours, but no COs was produced. After the GlcNAc was added, the COs was produced rapidly. The COs content reached 929 mg.L^-1 at 26 h. The overall productivity is 36 mg. L^-1.h^-1, while the productivity in one-step fermentation is only 25 mg.L^-1.h^-1.Biosynthesis of COs derivatives in large scale. GlcNAc derivatives of methyl-GlcNAc and allyl-GlcNAc were synthesized and added in the cultivation medium as precursors. Cultivation of recombinant E.coli (pCL3) in the presence of different precursor led to the biosynthesis of various COs derivatives. The intracellular COs products content, including COs and its derivatives, was 7.2% and 6.4% respectively, with methyl-GlcNAc and allyl-GlcNAc as precursor, which is higher than that of GlcNAc. The total COs yield was reached 913 mg. L^-1 and 801 mg. L^-1 in biosynthesis of allyl-COs and methyl-COs respectively. These GlcNAc derivatives, which are the most hydrophobic, are probably more efficiently bound by NodC.Confirmation of the structure of COs and its derivatives. Various COs components were purified after a series procedures, such as active charcoal adsorption, P4 gel filtration and preparative HPLC using an Cosmosil NH₂ 5μm, 250×10 mm HPLC column. The isolated products were analyzed by NMR and ESI-MS. The results confirmed the identification of the obtained samples as target tetraoside and pentaoside, respectively. ESI-MS data were shown as follows: penta-N-acetyl-chitopentaose: m/z, 1056 [M+Na]⁺, m/z, 1034 [M+H]⁺; tetra-N-acetyl-chitotetraose: m/z, 853 [M+Na]⁺; allyl-penta-N-acetyl-chitopentaose: m/z, 1096 [M+Na]⁺, m/z, 1074 [M+H]⁺; allyl-tetra-N-acetyl-chitotetraose: m/z, 893 [M+Na]⁺; methyl-tetra-N-acetyl-chitotetraose:m/z, 867 [M+Na]⁺, m/z, 845 [M+H]⁺; methyl-penta-N-acetyl-chitopentaose: m/z, 1070 [M+Na]⁺, m/z, 1048 [M+H]⁺. Overproduction and purification of recombinant NodB from E. coli. The T7promoter vector pET28a was used for expression of NodB_M and NodB_L proteins from S. meliloti and M. loti, respectively. Four recombinant proteins were overexpressed as inclusion bodies in LB cultures with induction of 0.5 mM IPTG The active NodB proteins were achieved through denaturation, refolding, affinity chromatograph and ultrafiltration. Enzyme assays were carried out in a final vol of 100 μl: 20 mM Mops (pH 7.2), 10 mg of substrate (penta-N-acetyl-chitopentaose), and 1 mg NodB protein. TLC results showed that fused NodB proteins with His-tag possess more deacetylase activity than NodB proteins fused with both of T7-tag and His-tag. ESI-MS result also confirmed the molecular weight of 991, indicating the structure of tetra-N-acetylchitopentaose.