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Journal of Bacteriology, February 2002, p. 821-830, Vol. 184, No. 3
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.3.821-830.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

Lisa Fontaine,1 Isabelle Meynial-Salles,1,{dagger} Laurence Girbal,1 Xinghong Yang,1 Christian Croux,1 and Philippe Soucaille1,2*

Centre de Bioingénierie Gilbert Durand, Laboratoire de Biotechnologies-Bioprocédés, UMR CNRS 5504, UR INRA 792, INSA, 31077 Toulouse cedex 4, France,1 Genencor International Inc., Palo Alto, California 943042

Received 10 April 2001/ Accepted 4 November 2001

The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.

* Corresponding author. Mailing address: Genencor International, 925 Page Mill Rd., Palo Alto, CA 94304. Phone: (650) 846-7696. Fax: (650) 845-6506. E-mail: psoucaille{at}genencor.com.

{dagger} CRITT-Bioindustries, INSA, DGBA, 31077 Toulouse cedex 4, France.

Journal of Bacteriology, February 2002, p. 821-830, Vol. 184, No. 3
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.3.821-830.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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