The Combination of Functional Metagenomics and an Oil-Fed Enrichment Strategy Revealed the Phylogenetic Diversity of Lipolytic Bacteria Overlooked by the Cultivation-Based Method

Abstract

Metagenomic screening and conventional cultivation have been used to exploit microbial lipolytic enzymes in nature. We used an indigenous forest soil (NS) and oil-fed enriched soil (OS) as microbial and genetic resources. Thirty-four strains (17 each) of lipolytic bacteria were isolated from the NS and OS microcosms. These isolates were classified into the (sub)phyla Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria, all of which are known to be the main microbial resources of commercially available lipolytic enzymes. Seven and 39 lipolytic enzymes were successfully retrieved from the metagenomic libraries of the NS and OS microcosms, respectively. The screening efficiency (a ratio of positive lipolytic clones to the total number of environmental clones) was markedly higher in the OS microcosm than in the NS microcosm. Moreover, metagenomic clones encoding the lipolytic enzymes associated with Alphaproteobacteria, Deltaproteobacteria, Acidobacteria, Armatimonadetes, and Planctomycetes and hitherto-uncultivated microbes were recovered from these libraries. The results of the present study indicate that functional metagenomics can be effectively used to capture as yet undiscovered lipolytic enzymes that have eluded the cultivation-based method, and these combined approaches may be able to provide an overview of lipolytic organisms potentially present in nature.

Isolation and characterization of a thermostable lipase from <i>Bacillus thermoamylovorans</i> NB501

“…Bacterial lipolytic enzymes were recently classified into eight different families according to their amino acid sequence (Arpigny and Jaeger, 1999). Recently, novel lipases were discovered using a metagenomics approach (Chow et al, 2012;Narihiro et al, 2014). In this study, we isolated a lipid-degrading thermophilic bacterium, Bacillus thermoamylovorans strain NB501.…”

Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

“…Instead, it is likely that tolerance to these compounds either requires the action of multiple, distal genes or is encoded by genes at low to intermediate abundance, such that the functional gene was not incorporated into the original metagenomic libraries. Alternative strategies (e.g., in situ enrichment of soil communities [44,45]) are likely required to expand both the number of compounds to which tolerance is observed and the diversity of tolerance-conferring genes recovered.…”

“…This outcome is likely a result of the incredible diversity of soil metagenomes, which confounds efforts to identify lowabundance genotypes. Methods to enrich these important yet lowabundance functions should improve the capture efficiency of functional selections and enable the discovery of new gene functions (44,45). Even without applying enrichment strategies, we discover numerous tolerance genes active against hemicelluloseand lignin-derived inhibitors, including genes of entirely unknown function (e.g., Fig.…”

The multi-omics promise in context: from sequence to microbial isolate

“…For example, inferences concerning the metabolic pathways for nitrogen and carbon cycling, respiration mechanisms and the degradation of particular toxic compounds can be based on the relative abundance or even the presence and absence of the relevant genes (Barone et al 2014;Narihiro et al 2014). With the constantly improved methods for sequence generation and bioinformatic analysis, near complete genome assembly is slowly becoming a standard method (Hug et al 2016).…”

“…These include soils (Dini-Andreote et al 2012;Fierer et al 2012), wastewater treatment bioreactors (Speth et al 2016), marine sediments (Plewniak et al 2013;Urich et al 2014) and eukaryotic host-associated microbiomes (Erickson et al 2012;Radax et al 2012;Sessitsch et al 2012;Segata et al 2013;Fuerst 2014), thereby rapidly increasing our knowledge and understanding of microorganisms and their key roles in biogeochemical cycling processes and eukaryotic host functioning and health. In addition, with metagenomics, whole genomes of newly discovered uncultured species can be resolved, allowing to predict the metabolic capabilities of these microorganisms in natural or man-made ecosystems (Tyson et al 2004;Siegl et al 2011;Hug et al 2012;Albertsen et al 2013;Wilson & Piel 2013;McLean et al 2013;Narihiro et al 2014;Nielsen et al 2014;Urich et al 2014;Walker et al 2014;Afshinnekoo et al 2015;Brown et al 2015).…”