Identification of a five‐oxidoreductase‐gene cluster from Acetobacter pasteurianus conferring ethanol‐dependent acidification in Escherichia coli

MicrobiologyOpen

Tamara Garcia-Armisen¹  Ken Vercammen¹  Tom Rimaux²  Gino Vrancken²  Luc De Vuyst² 

[1] Department of Bioengineering Sciences, Research group Microbiology, VIB Department of Structural Biology Vrije Universiteit BrusselPleinlaan 2, Brussels, Belgium;Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium

关键词: Central metabolism;    gene prediction;    gene transfer;    hydrogenases;   

DOI  :  10.1002/mbo3.4

来源: Wiley

【 摘 要 】

Abstract

Acetobacter pasteurianus, a Gram-negative bacterium belonging to the α-divison of Proteobacteria, produces acetic acid through ethanol oxidation. A genomic bank of A. pasteurianus 386B DNA was cloned in the low-copy cosmid pRG930Cm vector and the resulting clones were screened for the production of protease using the skimmed-milk agar assay whereby a clearing zone around the inoculated spots indicates casein degradation. Several positive clones were selected and restriction analysis revealed that many contained the same inserts. One clone was further analyzed and the cosmid DNA subjected to in vitro transposon insertion. After electroporation, several clones having lost the capacity to cause casein degradation were isolated and the sequence of the transposon-flanking regions analyzed. The majority of insertions mapped to one gene encoding an NAD(P)⁺-dependent aldehyde dehydrogenase (ALDH) of the PNTB superfamily, whereas one insert was found upstream in a gene encoding an ethanol dehydrogenase. Addition of phenol red to the medium confirmed the ethanol-dependent acidification around the inoculated spots of the clones without transposon insertion, suggesting that casein degradation is due to the production of acetic acid as a result of the combined activities of the alcohol dehydrogenase and ALDH. Quantitative data and pH measurements confirmed a significant acidification, and the presence of acetic acid.

【 授权许可】

CC BY-NC   
© 2012 The Authors. MicrobiologyOpen published by Blackwell Publishing Ltd.

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