Identification and Characterization of Coenzyme B 12 -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures
ABSTRACT To isolate genes encoding coenzyme B 12 -dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions....
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Published in | Applied and environmental microbiology Vol. 69; no. 6; pp. 3048 - 3060 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
01.06.2003
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Online Access | Get full text |
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Summary: | ABSTRACT
To isolate genes encoding coenzyme B
12
-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative
Escherichia coli
strain. In this way, two positive
E. coli
clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000
E. coli
clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of
Citrobacter freundii
and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from
E. coli
Bl21/pAK2.1 and
E. coli
Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from
E. coli
Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of
C. freundii
. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of
C. freundii
with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by
E. coli
Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol. |
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ISSN: | 0099-2240 1098-5336 |
DOI: | 10.1128/AEM.69.6.3048-3060.2003 |