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.
Highlights
As only a small fraction of all microorganisms from environmental samples can be isolated and studied by classic microbiological methods, culture-independent strategies, such as metagenomics, have been employed to overcome these restrictions (Torsvik et al, 1990; Amann et al, 1995; Handelsman et al, 1998)
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
Other hosts than E. coli have been shown to be good alternatives and new broad-range expression vectors have been developed for use in functional metagenomics (Courtois et al, 2003; Martinez et al, 2004; Aakvik et al, 2009; Craig et al, 2010; Damon et al, 2011; Kakirde et al, 2011; Kellner et al, 2011; Parachin and Gorwa-Grauslund, 2011; Liebl et al, 2014)
Summary
As only a small fraction of all microorganisms from environmental samples can be isolated and studied by classic microbiological methods, culture-independent strategies, such as metagenomics, have been employed to overcome these restrictions (Torsvik et al, 1990; Amann et al, 1995; Handelsman et al, 1998). Since only a fraction of the genes from random (meta)genomic gene libraries are expressed (Gabor et al, 2004; Sorek et al, 2007), the identification of truly novel genes from metagenomes is hampered when a single host is used (Jenney and Adams, 2008). Other hosts than E. coli (e.g., diverse Proteobacteria, Actinobacteria, and eukaryotic fungi) have been shown to be good alternatives and new broad-range expression vectors have been developed for use in functional metagenomics (Courtois et al, 2003; Martinez et al, 2004; Aakvik et al, 2009; Craig et al, 2010; Damon et al, 2011; Kakirde et al, 2011; Kellner et al, 2011; Parachin and Gorwa-Grauslund, 2011; Liebl et al, 2014)
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