Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

Ana Popović ,Tran Hai,Thomas Brüls,Alexei Savchenko,A Joachimiak ,Mahbod Hajighasemi,Peter N Golyshin,Denis Le Paslier,B Nocek ,Michail M Yakimov,T Skarina ,Robert Flick,Manuel Ferrer,Olga V Golyshina,Anatoly Tchigvintsev,Julia Glinos,Tatyana N Chernikova,V Yim ,Anna N Khusnutdinova,Greg Brown,Alexander F Yakunin

doi:10.1038/srep44103

Abstract

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.

Highlights

Is estimated that the global protein universe of microorganisms exceeds 1012 proteins indicating that we know astonishingly little about microbial proteins and enzymes[12,13]
Screening of purified proteins or metagenome gene libraries for enzymatic activity represents a direct experimental approach to identify the biochemical function of unknown proteins[5,7,20,21,22]
The metagenomic enzyme screening approach involves directly assaying proteins expressed from environmental DNA in a surrogate host for enzymatic activity against a specific chemical substrate[24]

Summary

Introduction

Is estimated that the global protein universe of microorganisms exceeds 1012 proteins indicating that we know astonishingly little about microbial proteins and enzymes[12,13]. Screening of purified proteins or metagenome gene libraries for enzymatic activity represents a direct experimental approach to identify the biochemical function of unknown proteins[5,7,20,21,22]. Most known carboxylesterases and lipases belong to the large protein superfamilies of α/β hydrolases and β-lactamases and have been classified into 16 families[36,37,38] Since these enzymes are of high interest for applications in biotechnology, a significant number of these proteins have been characterized both structurally and biochemically, mostly esterases from the α/β hydrolase superfamily[36,39,40,41]. The active site residues of new enzymes were identified using site-directed mutagenesis, and the crystal structure of a metal-dependent cyclase-like esterase provided insight into the molecular mechanisms of its activity

Methods

Results

Conclusion