title: Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus.
abstract: Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Leis
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Angel
authors.initials: A
authors.lastname: Angelov
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Mientus
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Haijuan
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Vu T T
authors.initials: VT
authors.lastname: Pham
authors.affiliation: Research Center Juelich, Institute of Bioorganic Chemistry, Heinrich-Heine-Universität Düsseldorf Juelich, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lauinger
authors.affiliation: Research Center Juelich, Institute of Bioorganic Chemistry, Heinrich-Heine-Universität Düsseldorf Juelich, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bongen
authors.affiliation: Research Center Juelich, Institute of Bioorganic Chemistry, Heinrich-Heine-Universität Düsseldorf Juelich, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Pietruszka
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Oeiras, Portugal.
authors.forename: Luís G
authors.initials: LG
authors.lastname: Gonçalves
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Oeiras, Portugal.
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
authors.affiliation: Department of Microbiology, Technische Universität München Freising, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liebl
keywords: Thermus thermophiles; comparative screening; functional metagenomics; novel metagenomic esterases; novel screening host