Sunday, July 26, 2009

Metabolic engineering of E. coli as biocatalyst producing D-psicose from fructose

Sook-Hee Lee1, Chong-Long Wang1, Asad Ali Shah1, Hui-Jeong Jang1, Jong-Wook Song1, Seo-hee Kang1, Min-Soo Ko2, Deok-Kun Oh3, and Seon-Won Kim1. (1) Division of Applied Life Science (BK21), EB-NCRC and PMBBRC, Gyeongsang National University, Jinju, 660-701, South Korea, (2) NanoHelix co. Ltd., Room 505, Taejeon Bioventure Town, 461-8 Jeonmin-dong Yuseong-gu, Daejeon, 305-348, South Korea, (3) Department of Bioscience and biotechnology, Konkuk University, Seoul, South Korea

D-Psicose, a rare keto-hexose, is not abundant in nature and is difficult to prepare by chemical methods. D-Psicose has sweet taste like general sugars, but calorie is near to zero because it is not metabolized in the human body. D-Psicose is 3-epimer of D-fructose which is abundant and relatively cheap. Psicose can be produced biotechnologically from epimerization of fructose. Enzymes for the 3-epimerization between fructose and psicose catalyze the sugars of either phosphorylated or nonphosphorylated state. E. coli has psicose-6-phosphate 3-epimerase (encoded by alsE) with substrate specificity to phosphorylated sugars. The alsE participates in allose metabolic pathway and is located in allose metabolic operon. However, E. coli alsE is not appropriate for psicose production because its reaction requires phosphorylation of fructose and dephosphorylation of psicose-6-phosphate. For the production of D-psicose from E. coli, we used D-psicose 3-epimerase (encoded by dpe) of Agrobacterium tumefeciens, which isomerizes fructose to psicose. E. coli as workhorse for psicose production was further engineered by deletion of pfkA and allose metabolic operon (als). The deletion of pfkA encoding 6-phosphofructokinase I could reduce fructose consumption via glycolysis and the deletion of allose operon, catabolic degradation of psicose through allose metabolic pathway. E. coli MG1655 (ΔpfkA, Δals) harboring pTPE plasmid for overexpression of dpe produced 150g/L of psicose from 80% (w/v) of fructose in 48 hours of whole cell conversion. This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, EB-NCRC (Grant No. R15-2003-012-02001-0), and BK21 program of Korea.