Co-expression of
Triosephosphate Isomerase, Fructose-1,
6-bisphosphate Aldolase and Fructose-1,6-bisphosphatase in E.coli
TANG Gong-Li, YANG Chun-Song, BAO Jian-Shao, WANG Yan-Fang,
CHEN Hai-Bao*
( State key Laboratory of Bio organic and Natural Products Chemistry,
Shanghai Institute of Organic Chemistry, the Chinese Academy of Sciences,
Shanghai 200032, China )
SHI Ding-Ji, LIU Feng-Long
( Laboratory of Photosynthesis, Institute of Botany, the Chinese Academy of
Sciences, Beijing 100093, China )
Abstract To
establish a way to control or to decrease the daily increasing concentration of
atmospheric CO2, metabolically engineering Cyanobacteria
was taken for the improvement of its efficiency of photosynthetic CO2
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 μmol 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.
Key words triosephosphate isomerase；fructose-1,6-bisphosphate
aldolase；fructose-1,
6-bisphosphatase；co-expression
*Corresponding author：Tel,86-21-64163300；Fax,86-21-64166128；e-mail,[email protected]