Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.
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 beta-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 beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-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 beta-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 beta-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 beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.
Tummala
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SB
Department of Chemical Engineering, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Welker
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NE
Papoutsakis
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ET
D000101:Acetyl-CoA C-Acetyltransferase / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D017930:Genes, Reporter; D006863:Hydrogen-Ion Concentration; D007763:Lac Operon; D010709:Phosphate Acetyltransferase / Q000235:genetics; D011401:Promoter Regions, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D010709:Phosphate Acetyltransferase; D000101:Acetyl-CoA C-Acetyltransferase; D001616:beta-Galactosidase; D002262:Carboxy-Lyases; C023743:acetoacetate decarboxylase