Cloning and expression of xylonate dehydratase from Caulobacter vibrioides

Butanetriol dehydratase Escherichia coliAim: Lignocellulosic biomass such as agricultural wastes (corn stover, sugar beet pulp and citrus peel) is a widely abundant and attractive source for the production of biofuels and chemicals.biofuels are sources of clean and renewable energy that are considered as a potential substitute for non-renewable oil fuels.various methods and processes have been tested by scientists and researchers in this field and the most favorable conditions for producing biofuels from biomass.however, this biomass has not been fully exploited in many parts of the world for biofuel production, especially in developing countries, and there is little relation with crop residues and forest and waste in this area.so much work is still needed to replace fossil fuels with biofuels from biomass.lignocellulosic waste biomass such as cassava peels, sugar beet pulp, and Ulva lactuca are suitable materials for bioethanol synthesis.D-xylose, is the second most abundant sugar in lignocellulosic hydrolysates.Nowadays, considerable efforts have been made to expand microbial cell factories to use D-xylose for the production of value-added chemicals.D-1,2,4-butanetriol (BT) is an extremely important intermediate chemical, is widely used in many fields, such as pharmaceuticals, paper, polymer materials, and military applications.A molecule 1,2,4-butanteriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT(the R-enantiomer).BT is widely used in the military industry, medicine, tobacco, polymer.A synthetic pathway involving four enzymes-D-xylose dehydrogenase (XDH), D-xylonate dehydratase (XD), 2-keto acid decarboxylase (KDC), and aldehyde reductase (ALR)-has been proposed and implemented to produce BT from D-xylose, highlighting its significant role in bioproduction.And because in most studies, the xylonate dehydratase was used in the case of Caulobacter.crescentus,and given the very high genetic similarity between Caulobacter.crescentusand Caulobacter.vibrioides, the aim of this study is to clone and express xylonate dehydratase gene of C.vibrioides in the E.coli.For this purpose, the research was carried out with the aforementioned methods.Material and methods: The xylonate dehydratase gene was retrieved from the NCBI database and amplified using PCR with specific primers after extracting the C. vibrioides genome.The piece of the gene was cloned in the pET28 expression vector and then transferred to the E.coli prepared cells using chemical methods.After the induction of the cells, recombinant protein expression was examined using SDS-PAGE.Results: By using restriction enzymes, Colony PCR and sequencing, the cloning process and the entry of the gene into the pET28 expression vector was confirmed.