Genes Cloning And Analysis Of Starch Synthesis Pathway In Thermophilic Caldicellulosiruptor Bescii

Cellulose is one of the most abundant reproducible material which is annuallyproduced up to1550billion tons by photosynthesis plants, among these hemicelluloseis about85billion tons. The energy release of all these ematerial would provide tentimes more than the total energy consumption in the world. However the processedcellulose annually only about2%of the total amount nowadays. A lot of cellulose isbeing wasted. By biochemistry and molecular biology technique, the cellulose can beconverted to glycogen, which has important implications for the use of naturalresources as cellulose. Thermophiles Caldicellulosiruptor bescii DSM6725can use avariety of fiber as carbon source, such as cellulose, and create mono-orpolysaccharide. The thermophiles genome had been analyzed and the metabolicpathway is relatively clear, which will facilitate the study of metabolic pathway ofthermophiles. With bioinformatics analysis, we found that thisbacteria contains aglycogen synthesis pathway, which probably could use cellulose as raw material forsynthesis of glycogen.We first analyze the protein sequences of glycosyl transferases, glycogensynthase and cellobiose phosphorylase of Caldicellulosiruptor bescii DSM6725.predict their functionand also built structure model for these enzymes. Wefound outthe related key amino acid residues for activition. We cloned the gene of glycogensynthase (Athe-0555), two isoforms of glycosyl transferase which is also called ADP-glucose phosphorylase (Athe-0556, the Athe-0557), and cellobiose phosphorylase(Athe-xl). These four proteins were also expressed in E. Coli and purified withaffinity purification. The recombinant protein Athe-0557and Athe-xl could bepurified but not forthe recombinant protein Athe-0555, Athe-0556. New methods iscarried out for effective purification of those two protein.In summary, with four cloned genes encoding starch synthesis enzymes, twopurified proteins, and predicted enzyme model via bioinformatics analysis, we have primarily understood several enzymes that were essential for starch synthesis inCaldicellulosiruptor bescii DSM6725. This study set up the basic knowledge forrelated research.