Tang Gong-Li, Yang Chun-Song, Bao Jian-Shao, Wang Yan-Fang, Chen Hai-Bao, Shi Ding-Ji, Liu Feng-Long
State key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, the Chinese Academy of Sciences, Shanghai 200032, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2001;33(1):131-136.
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.
为建立一种控制或降低大气中二氧化碳日增浓度的方法，人们采用对蓝藻进行代谢工程改造的方式来提高其光合固定二氧化碳的效率。作为本研究的初步阶段，分别编码三种重要卡尔文循环酶（即磷酸丙糖异构酶（TPI）、果糖-1,6-二磷酸醛缩酶（FBP醛缩酶）和果糖-1,6-二磷酸酶（FBP酶））的三个基因已被克隆到一个由trc启动子控制的质粒pTrcFAT中。这三个基因的成功共转录表达导致在0.25 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导下这些酶产量很高。生物测定表明，来自一升培养物的表达酶每分钟可直接催化二羟基丙酮磷酸（DHAP）转化为700 μmol的果糖-6-磷酸（F-6-P）。此外，为了将这三个基因共表达系统引入蓝藻，利用质粒pTrcFAT和穿梭载体pDC-8构建了一种大肠杆菌和蓝藻之间的穿梭质粒，形成了一个含有三个基因共表达操纵子二聚体的穿梭质粒pDCFAT-2。已在大肠杆菌中成功共表达了具有更高全活性的pDCFAT-2。该穿梭质粒用于转化蓝藻聚球藻属PCC 7942，并获得了一些阳性菌落。
