A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes

Abstract

Background: 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). 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.

Results: Here, 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.

Conclusions: This 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.

Fig. 1

Schematic representation of the methodology used to screen for (hemi) cellulases in fosmid-based metagenomic libraries. 1) Biased communities (e.g. soil-derived microbial consortia cultivated on wheat straw) are at the basis of enhanced hit rates. 2) Screening of fosmid pools (700 fosmids per pool) allow high-throughput analyses. 3) Screening on substrate mixtures accelerate the analyses. 4) Re-screening of positive clones in single, double and further mixed combinations of substrates enable the detection of specific activities

Fig. 2

Graphical representation of the genes detected and annotated in seven fosmid inserts selected by the multi-substrate screening approach

Fig. 3

Functional analyses (relative enzymatic activities, expressed in U/mg) based on total proteins extracted from selected positive fosmid clones. a Characterization of the fosmid clones using three pNP-substrates (pNPGal, pNPXyl and pNPGlu). b and c Effect of temperature and pH on the activity of clones NT2-2 and T17-2 with pNPGal. d and e Effect of temperature and pH on the activity of clones T5-5, T4-1 and 10BT with pNPXyl

Fig. 4

Relative enzymatic activities of the total proteins produced from the fosmid clones NT2-2, T5-5 and NT18-17 using pNPGal, pNPXyl and pNPGlu, respectively, at different temperatures and pH values. a and b Effect of temperatures and pH on the activity of fosmid clone NT2-2. c and d Effect of temperatures and pH values on the activity of the fosmid clone T5-5. e and f Effect of temperature and pH on the activity of fosmid clone NT18-17

Fig. 5

Native polyacrylamide gel electrophoresis (Native-PAGE) and zymogram analysis. Left: Native polyacrylamide gel of the total proteins from the positive fosmid clones. Right: Positive fosmid clones that showed bands (red square) with activity on the zymogram assay using MUFGal (MUF-beta-D-galactopyranoside) and MUFXyl (MUF-beta-D-xylopyranoside) as substrates