title: Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.
abstract: To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained.
authors.affiliation: State key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, the Chinese Academy of Sciences, Shanghai 200032, China. hbchen@pub.sioc.ac.cn
authors.forename: Gong-Li
authors.initials: GL
authors.lastname: Tang
authors.forename: Chun-Song
authors.initials: CS
authors.lastname: Yang
authors.forename: Jian-Shao
authors.initials: JS
authors.lastname: Bao
authors.forename: Yan-Fang
authors.initials: YF
authors.lastname: Wang
authors.forename: Hai-Bao
authors.initials: HB
authors.lastname: Chen
authors.forename: Ding-Ji
authors.initials: DJ
authors.lastname: Shi
authors.forename: Feng-Long
authors.initials: FL
authors.lastname: Liu