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.
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.
Fontaine
Lisa
L
Centre de Bioingénierie Gilbert Durand, Laboratoire de Biotechnologies-Bioprocédés, UMR CNRS 5504, UR INRA 792, INSA, 31077 Toulouse cedex 4, France.
Meynial-Salles
Isabelle
I
Girbal
Laurence
L
Yang
Xinghong
X
Croux
Christian
C
Soucaille
Philippe
P
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D000440:Butanols / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D000440:Butanols; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009243:NAD; D000431:Ethanol; C446050:AdhE2 protein, Clostridium acetobutylicum; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C060833:butanol dehydrogenase; D000426:Alcohol Dehydrogenase; C068383:adhE protein, E coli; D000445:Aldehyde Oxidoreductases; C056375:butyraldehyde dehydrogenase (Coenzyme A-linked)