Preliminary Study On Key Enzymes For Metabolism Of Glucose Metabolism In Escherichia Coli BL21 (DE3)

Glucosamine, GlcN,2-amino-2-deoxy-D-glucose, an important amino sugar, it is component of mucopolysaccharide and chitin. For mammals, glucosamine is essential ingredient of mucosal secretions, connective tissue, skin, tendons, ligaments and cartilage. Osteoarthritis, which is a common disease in the elders, is a degeneration of articular cartilage causing pain and joint dysfunction (including joint deformity).Glucosamine, a widely used nutritional supplement, has a content efficacy of osteoarthritis. Other studies also showed that any dose of oral glucosamine make little effect on fasting glucose levels, glucose metabolism and insulin sensitivity. There were clear evidences that normal dose of oral glucosamine cannot induce the development of type II diabetes.L-glutamine:D-fructose-6-phosphate aminotransferase, also known as Glucosamine-6-phosphate synthase (GlcN-6-P Synthase, EC2.6.1.16). It catalyzed the reaction of Fru-6-P to GlcN-6-P, with L-glutamine (Gln) as a nitrogen donor, the equation is as follows:D-fructose-6-phosphate+L-glutamineâ†'L-glutamate+D-glucosamine-6-phosphateIt is the first and rate-limiting step of biosynthesis of UDP-GlcNAc. So is the core of metabolic regulation of glucosamine macromolecules. This biochemical response is the only pathway of the biosynthesis of GlcN as we knowm till now.Metabolic engineering is the engineering that optimize the cell competent to increase the cell substance production through the modification of the regulatory process. In the light of the metabolic engineering strategies, a strain which has a glucosamine production performance was constructed. At first step, a λ phage Red homologous recombination system was use to delete key genes relate GlcN regulation: mannose-specific-phosphotransferase system (manXYZ), acetyl glucosamine-specific transporter and metabolic enzyme systems (including phosphotransferase, deacetylase and deaminase:nagBACD-nagE). The growth curve of YP1103and the wild type strain in different media showed that YP1103cannot use GlcN and GlcNAc as the only carbon source; the ability of Glc usage is also decreased. This experiment not only proved the completion of knocking out the two operons, but also verified the property of strains with GlcN and GlcNAc enrichment. On the other hand, we modified the glucosamine synthase gene (glmS) of E. coli BL21(DE3) through directed evolution. A6×His tag is inserted in the middle at the position225threonine. An expression vector pETG was constructed and its soluble expression in YP1103was achieved. After Ni-NTA affinity chromatography step to purify GlmS protein, an8.63unit/mg enzyme was acquired, consistent with other studies reported.In summary, E. coli BL21(DE3) was successful reconstructed to provide the initial characteristics of the ideal glucosamine-producing strain. This work was a groundbreaking exploration for the further transformation of the suitable strain for industrial production.