Sunday, May 3, 2009
2-49

Construction of a reporter gene system for use in the ethanologen Geobacillus thermoglucosidasius SB2

Jeremy Bartosiak-Jentys and David J. Leak. Biology, Imperial College London, Room 326 Biochemistry Building, Exhibition Road, London, SW7 2AZ, United Kingdom

The thermophilic bacterium, Geobacillus thermoglucosidasius SB2 is a facultative anaerobe that ferments a range of C5 and C6 sugars making it an attractive candidate for bioethanol production. Fermentation of D-glucose by G. thermoglucosidasius SB2 results in mixed acid production (ethanol, L-lactate, acetate and formate). Production of unwanted fermentation products can be prevented by creating strains where genes, encoding key enzymes in the synthesis pathways of these acids, have been knocked out. However, for increased carbon flux to ethanol the upregulation of key genes (e.g. the pdh operon) under fermentative conditions is advantageous. Increased expression can be achieved by inserting a promoter active under the desired conditions upstream of the gene to be upregualted.

In order to asses promoter strength, a reporter gene plasmid containing the pheB gene from Geobacillus stearothemophilus DMZ6285, which encodes a thermophilic catechol 2,3 dioxygenase (C23O), was constructed in the Escherichia coli ­– G. thermoglucosidasius shuttle vector pUCG18. Six sequentially shorter fragments of the pdhAα upstream region, based on in silico predicted promoter sequences, were cloned 5’ to the pheB gene. C23O enzyme assays and qRTPCR data indicates all promoter fragments were able to initiate transcription of pheB under aerobic conditions and at a reduced level in micro-aerobic cultures. However, fragments int1 and int2, which correspond to the promoter sequences proximal to the pdhAα gene, were also found to allow expression under anaerobic conditions. This system could be used to elucidate a promoter sequence that allows constitutively high expression of its downstream gene under all growth conditions.


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See more of The 31st Symposium on Biotechnology for Fuels and Chemicals (May 3-6, 2009)