Abstract
BackgroundFunctional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered.ResultsHere, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90 % of enzymatic activity at 55 °C and pH 10.0.ConclusionsThis study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2404-0) contains supplementary material, which is available to authorized users.
Highlights
Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes
Two different strategies can be used: i) the unleashing of high-throughput DNA sequencing on degradative consortia, and/or ii) the selection of enzymes via functional/genetic screening of metagenomic libraries produced from these consortia [9]
Construction and functional screening of two fosmid metagenomic libraries Two metagenomic libraries were produced in fosmids, one from pooled raw wheat straw (RWS) consortial DNA (~70,000 clones) and another one from torrified wheat straw (TWS; ~70,000 clones)
Summary
Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). The cellulose moiety is a glucose polymer, whereas the hemicellulose part is composed of various pentose and hexose sugars (e.g. xylose, arabinose, mannose and galactose) linked by beta/alpha-glycosidic bonds [2,3,4] All of these sugars have great value for the production of bioethanol, biodiesel and/or plastics [5, 6], and so there have been many efforts to release them from the plant matrix. Current physicochemical methodologies for the degradation of plant biomass and subsequent production of sugars are imperfect [7] and so there is a great interest in the development of alternative and efficient processes, based on enzymes and/or lignocellulolytic microbes [8, 9]. Two different strategies can be used: i) the unleashing of high-throughput DNA sequencing on degradative consortia, and/or ii) the selection of enzymes via functional/genetic screening of metagenomic libraries produced from these consortia [9]
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