Construction of an Expression Vector Harboring Xylonate Dehydratase Enzyme Gene and its Transformation into the Appropriate Host

Lignocellulosic biomass such as agricultural wastes are important and available source for production of biofuels and chemicals.D-xylose, is the second important sugar in lignocellulosic hydrolysates .Nowadays, developing the microbial cell factories is important to acheive D-xylose for the production of chemicals.D-1,2,4butanetriol (BT), an important intermediate chemical, apply in many fields, such as pharmaceuticals, polymer materials, paper and military.Bio production of BT is necessary, so a synthetic pathway (using D-xylose dehydrogenase (XD), D-xylonate dehydratase, 2-keto acid decarboxylase and aldehyde reductase) applied to produce BT from D-xylose.XD have Fe-S clusters so its belongs to the IlvD/EDD dehydratase enzymes.Crystallographic analysis demonstrated that XylD has a homotetrameric quaternary structure, and each monomer is composed of two domains.XD catalyzes conversion of D-xylonic acid to 2-keto-3-deoxy-D-xylonic acid (KDX).In this research by using restriction enzymes, Colony PCR and sequencing, the cloning process and entry of the gene xylD into the expression vector pET28 were confirmed.Existence of recombinant protein was verified by SDS-PAGE with the molecular weight of about 68 KDa.The expression rate of the recombinant protein was estimated to be 54% by Image J software.Therefore, the bioproduction of butanetriol requires the construction of a metabolic pathway consisting of several enzymes.The presence of E.coli bacteria as the target strain and using a cheap substrate such as biomass containing D-xylose and the presence of the xylonate dehydratase enzyme are necessary to produce BT at a high rate.