Process and Materials for Production of Glucosamine
a technology of glucosamine and process, applied in the direction of esterified saccharide compounds, sugar derivates, transferases, etc., can solve the problems of increasing the availability of raw materials, reducing affecting the production efficiency of chitin, etc., to increase the action of phosphatase and increase the action of glucosamine-6-phosphate synthase
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example 1
[0125]The following example describes the production of mutant Escherichia coli strains which are blocked in amino acid sugar metabolic pathways involving degradation of glucosamine.
[0126]The starting strain for the construction of all glucosamine overproducing strains described herein was E. coli W3110 (publicly available from the American Type Culture Collection as ATCC No. 25947), a prototrophic, F−λ− derivative of E. coli K-12 (Bachmann, 1987, “Escherichia coli and Salmonella typhimurium”, Cellular and Molecular Biology, pp. 1190-1219; incorporated herein by reference in its entirety). A list of all strains used and produced in the following examples is provided in Table 1.
TABLE 1Bacterial strains.StrainAliasGenotypeSource / ReferenceW3110F− mcrA mcrB IN(rrnD-rrnE) 1 λ−ATCCIBPC 522thi-1 argG6 argE3 his-4 mtl-1 xyl-5 rpsLJ. Plumbridgetsx-29? ΔlacX74 manXYZ8 nagE47ptsG22 zcf-229::Tn10IBPC 566thi-1 argG6 argE3 his-4 mtl-1 xyl-5 rpsLJ. Plumbridgetsx-29? ΔlacX74 manXYZ8 zdj-225::Tn10IB...
example 2
[0135]The following Example describes the cloning and overexpression of the glmS gene and the integration of the T7-glmS gene cassette into the E. coli chromosome.
[0136]Cloning and Overexpression of the glmS Gene.
[0137]Using information obtained from the published sequence of the glmS gene (Walker et al., 1984, Biochem. J., 224:799-815, which is incorporated herein by reference in its entirety), primers were synthesized to amplify the gene from genomic DNA isolated from strain W3110 (Table 1) using the polymerase chain reaction (PCR). The primers used for PCR amplification were designated Up1 and Lo8 and had the following sequences:
(SEQ ID NO: 1)Up1:5′-CGGTCTCCCATGTGTGGAATTGTTGGCGC-3′(SEQ ID NO: 2)Lo8:5′-CTCTAGAGCGTTGATATTCAGTCAATTACAAACA-3′
[0138]The Up1 primer contained sequences corresponding to the first twenty nucleotides of the glmS gene (represented in nucleotides 10-29 of SEQ ID NO:1) preceded by a BsaI restriction endonuclease recognition site (GGTCTC, represented in nucleot...
example 3
[0151]The following example shows the effect of strain genotype on glucosamine accumulation.
[0152]Strains with T7-glmS integrants, produced as described in Example 2, as well as the corresponding parent strains without integrated DNA, were grown in shake flasks containing M9A medium (14 g / L K2HPO4, 16 g / L KH2PO4, 1 g / L Na3Citrate-2H2O, 5 g / L (NH4)2SO4, pH 7.0) supplemented with 20 g / L glucose, 10 mM MgSO4, 1 mM CaCl2, and 1 mM IPTG. Samples were taken periodically over the course of two days, and the glucosamine concentration in the culture supernatant was measured using the modified Elson-Morgan assay as described in Example 2. Samples were assayed with and without acetic anhydride treatment, and the amount of glucosamine present was determined from the net absorbance using a standard curve.
[0153]Glucosamine concentrations after 24 hours of cultivation, at which time the concentration peaked, are indicated in Table 3. The results shown in Table 3 indicate that for significant gluco...
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