Abstract
A gene expression reporter system (pHT3) for
Clostridium acetobutylicum
ATCC 824 was developed by using the
lacZ
gene from
Thermoanaerobacterium thermosulfurogenes
EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes,
ptb
(coding for phosphotransbutyrylase),
thl
(coding for thiolase), and
adc
(coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in
C. acetobutylicum
. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the
lacZ
gene from
T. thermosulfurogenes
EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the
ptb
promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.
