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 dehydratasecontaining 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.

Use of Glycerol in Biotechnological Applications

“…Purified 1,3-propanediol dehydrogenase from E. agglomerans CNCM 1210 was shown to be inhibited by NAD + (K i 0.29 mM) and 1,3-PDO (K i 13.7 mM) and therefore might be limiting production yields of 1,3-PDO (Barbirato et al, 1997). Also the glycerol dehydratases from C. freundii and metagenome samples were shown to be inhibited by 1,3-PDO (Knietsch et al, 2003), moreover glycerol dehydratases from K. pneumoniae and C. freundii are inhibited by deactivation by glycerol (Tobimatsu et al, 1999, Tobimatsu et al, 2000, Kajiura et al, 2001, Seifert et al, 2001. To overcome production limitations by e.g.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…The most common and conventional nucleotide sequence-based approaches are PCR amplification of target gene(s) or oligonucleotide probe-based hybridization experiments (Knietsch, Bowien et al 2003, Daniel 2005, Lawley and Tannock 2012. PCR has been used extensively to retrieve partial sequences of target genes for both taxonomic diversity as well as functional studies (Lorenz, Liebeton et al 2002, Cowan, Meyer et al 2005).…”

“…The metagenomic clone that complements the desired function in the mutant strain is isolated and sequenced for the identification of coding gene(s) (Wang, Meek et al 2006). In the functional complementation of a constructed dehydratasenegative Escherichia coli strain, two positives were obtained out of 560,000 tested clones (Knietsch, Bowien et al 2003).…”

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

“…To help the understanding of the role of each subunit in the catalysis, a study of the hybrid enzymes between diol and glycerol dehydratases may be useful, but not yet reported so far. Complementation of incompletely cloned diol dehydratase-or glycerol dehydratase-encoding gene regions from environmentally derived bacteria with the respective enzyme genes of known bacteria was reported ( 26 ).…”