Cloning and Expression of Xylose Dehydrogenase in E.Coli

Background and aim: as we know, by manipulating the genes of microorganisms, metabolic pathways can be designed to produce different substances like xylonic acid using in the production of copolyamides, polyesters and hydrogels. xylonic acid is a five-carbon sugar acid that is formed by xylose dehydrogenase (xdh) during the oxidation of xylose. this enzyme, consist of a homotetramer protein and its activity is dependent on nad(p) as a co-substrate, converts xylose to xylonolactone, an intermediate compound containing an aromatic ring, then the lactone is cleaved spontaneously or by lactonase and finally xylonic acid is formed. several species of bacteria, pseudomonas, acetobacter, aerobacter, gluconobacter, erwinia, oxidize xylose to xylonic acid. xylonic acid is structurally similar to gluconic acid and has a same application although it is inexpensive and derived of the non-food hemicellulose. so this sugar is the best alternative in production of industrial materials such as food products, solvents, adhesives, dyes, paints and polishes. with high production and purification of xylonate, it can be used as a precursor of pharmaceutical and chemical substances such as 1,2,4butanetriol. also xylonate can be converted to other sugars like gluconate (as a sweetener in the food industry). in addition xylose dehydrogenase can be used as a biosensor to detect xylose and identify the metabolic characteristics of new discovered organism.methods: the gene of xylose dehydrogenase was amplified by specific primers from extracted genome of caulobacter vibrioides and by enzymatic cleavage in gene and vector cloning was performed. eventually the presence of genes in recombinant vector was confirmed by pcr and digestion.results: in order to produce xylonic acid the species of bacteria was engineered containing the gene of xylose dehydrogenase (xdh) and lactonase (xylc). due to the lack of xdh in e.coli, it cannot produce xylonic acid. thus, by transforming and expressing the gene of these enzymes it can convert xylose to xylonate with high-efficiency. conclusion: cloning and transforming xylose dehydrogenase into e.coli was performed. subsequently we will analyze presence of xylonic acid into culture media and detect this conversion and analyze its efficiency and property of transformation in e.coli. we hope to achieve high xylonic acid production by eliminating competitive pathways and optimizing this strain.