The distribution of novel bacterial laccases in alpine paleosols is directly related to soil stratigraphy.

Bacterial laccases are now known to be abundant in soil and to function outside of the cell facilitating the bacterial degradation of lignin. In this study we wanted to test the hypotheses that: i) Such enzymes can be identified readily in stratified paleosols using metagenomics approaches, ii) The distribution of these genes as potential 'public good' proteins in soil is a function of the soil environment, iii) Such laccase genes can be readily retrieved and expressed in E. coli cloning systems to demonstrate that de novo assembly processes can be used to obtain similar metagenome-derived enzyme activities. To test these hypotheses, in silico gene-targeted assembly was employed to identify genes encoding novel type B two-domain bacterial laccases from alpine soil metagenomes sequenced on an Illumina MiSeq sequencer. The genes obtained from different strata were heterologously cloned, expressed and the gene products were shown to be active against two classical laccase substrates. The use of a metagenome-driven pipeline to obtain such active biocatalysts has demonstrated the potential for gene mining to be applied systematically for the discovery of such enzymes. These data ultimately further demonstrate the application of soil pedology methods to environmental enzyme discovery. As an interdisciplinary effort, we can now establish that paleosols can serve as a useful source of novel biocatalytic enzymes for various applications. We also, for the first time, link soil stratigraphy to enzyme profiling for widespread functional gene activity in paleosols.