Date Published: September 29, 2017
Publisher: Springer Berlin Heidelberg
Author(s): Anna Lewin, Jinglie Zhou, Vu Thuy Trang Pham, Tone Haugen, Mohamed El Zeiny, Olav Aarstad, Wolfgang Liebl, Alexander Wentzel, Mark R. Liles.
Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.
Extremophiles have attracted great interest for their ability to thrive in conditions that sometimes include multiple environmental extremes and for their enzymes that have adapted to these extreme conditions and therefore may have industrial applications (Rothschild and Mancinelli 2001). Taq DNA polymerase is the classic example of an enzyme from a thermophile, i.e. Thermus aquaticus, isolated in culture from hot springs at Yellowstone National Park (Chien et al. 1976). The traditional approach used to identify and exploit these enzymes is culture-dependent, which greatly restricts the accessible diversity of thermophile-derived natural products.
The results of the present study confirm our earlier analyses that the processed oil reservoir sample was dominated by members of the domain Archaea, phylum Euryarchaeota (Kotlar et al. 2011; Lewin et al. 2014). The results obtained from direct 454 pyrosequencing and from the Illumina sequencing of the fosmid library were highly comparable in terms of phylogenetic and functional composition (Fig. 1a, b). The alpha-diversity of the shotgun sequences database as determined based on Shannon’s Diversity Index was 42.08 species, which is relatively low compared to non-extreme environments. Despite of a few exceptions, the overall high consistency of the sequencing results indicates that overall biases are relatively low. Given the observation that the Archaea are dominant in this environment and that most of the enzymes obtained in this study were predicted to be derived from taxa affiliated with the Euryarchaeota, we conclude that our sampling of these oil reservoir microbial assemblages has been inclusive of much of the extant phylogenetic and functional diversity. The large number of unassigned sequences from both direct and library sequencing indicates that even though this is an extreme habitat with limited phylogenetic breadth, there is a considerable amount of previously unknown metagenomic diversity in the sampled environment.