title: Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade.
abstract: Sugar phosphates cannot be produced easily by microbial fermentation because negatively-charged compounds cannot be secreted across intact cell membrane. D-xylulose 5-phosphate (Xu5P), a very expensive sugar phosphate, was synthesized from D-xylose and polyphosphate catalyzed by enzyme cascades in one pot. The synthetic enzymatic pathway comprised of xylose isomerase and xylulokinase was designed to produce Xu5P, along with a third enzyme, polyphosphate kinase, responsible for in site ATP regeneration. Due to the promiscuous activity of the ATP-based xylulokinase from a hyperthermophilic bacterium Thermotoga maritima on polyphosphate, the number of enzymes in the pathway was minimized to two without polyphosphate kinase. The reactions catalyzed by the two-enzyme and three-enzyme pathways were compared for Xu5P production, and the reaction conditions were optimized by examining effects of reaction temperature, enzyme ratio and substrate concentration. The optimized two-enzyme system produced 32 mM Xu5P from 50 mM xylose and polyphosphate after 36 h at 45°C. Biosynthesis of less costly Xu5P from D-xylose and polyphosphate could be highly feasible via this minimized two-enzyme pathway.
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, 24061, Virginia.
authors.forename: Jae-Eung
authors.initials: JE
authors.lastname: Kim
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, 24061, Virginia. ypzhang@vt.edu.|Cell Free Bioinnovations Inc., Blacksburg, Virginia. ypzhang@vt.edu.|Institute for Critical Technology and Applied Science (ICTAS), Virginia Tech, Blacksburg, Virginia. ypzhang@vt.edu.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China. ypzhang@vt.edu.
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D010428:Pentosephosphates / Q000096:biosynthesis*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology; D014994:Xylose / Q000378:metabolism*
chemical_list: D004798:Enzymes; D010428:Pentosephosphates; D011122:Polyphosphates; C031625:xylulose-5-phosphate; D000255:Adenosine Triphosphate; D014994:Xylose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
keywords: ATP regeneration; D-xylulose 5-phosphate; in vitro synthetic biosystem; sugar phosphate; synthetic enzymatic pathway; xylose