Heterologous expression of clostridial hydrogenase in the Cyanobacterium synechococcus PCC7942.
The Clostridium pasteurianum hydrogenase I has been expressed in the cyanobacterium Synechococcus PCC7942. The Shine-Dalgarno sequence of the structural gene encoding hydrogenase I from C. pasteurianum was changed to that of the cat (chloramphenicol acetyltransferase) gene. The hydrogenase gene was cloned downstream of a strong promoter, isolated from Synechococcus PCC7942, with the cat gene as a reporter gene. Expression of clostridial hydrogenase was confirmed by Western and Northern blot analyses in Synechococcus and Escherichia coli, whereas in vivo/in vitro measurements and activity staining of soluble proteins separated on non-denaturing polyacrylamide gels revealed functional expression of hydrogenase only in cyanobacterial cells. The changed Shine-Dalgarno sequence appeared to be essential for the functional expression of clostridial hydrogenase in Synechococcus, but had no influence on the expression and activity of clostridial hydrogenase expressed in E. coli.
Asada
Y
Y
Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama-shi, Japan.
Koike
Y
Y
Schnackenberg
J
J
Miyake
M
M
Uemura
I
I
Miyake
J
J
D015152:Blotting, Northern; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D000458:Cyanobacteria / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005796:Genes; D005798:Genes, Bacterial; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids
D006864:Hydrogenase; D015500:Chloramphenicol O-Acetyltransferase