Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community

Abstract

Background: Degradation of pectin in lignocellulosic materials is one of the key steps for biofuel production. Biological hydrolysis of pectin, i.e., degradation by pectinolytic microbes and enzymes, is an attractive paradigm because of its obvious advantages, such as environmentally friendly procedures, low in energy demand for lignin removal, and the possibility to be integrated in consolidated process. In this study, a metagenomics sequence-guided strategy coupled with enrichment culture technique was used to facilitate targeted discovery of pectinolytic microbes and enzymes. An apple pomace-adapted compost (APAC) habitat was constructed to boost the enrichment of pectinolytic microorganisms.

Results: Analyses of 16S rDNA high-throughput sequencing revealed that microbial communities changed dramatically during composting with some bacterial populations being greatly enriched. Metagenomics data showed that apple pomace-adapted compost microbial community (APACMC) was dominated by Proteobacteria and Bacteroidetes. Functional analysis and carbohydrate-active enzyme profiles confirmed that APACMC had been successfully enriched for the targeted functions. Among the 1756 putative genes encoding pectinolytic enzymes, 129 were predicted as novel (with an identity <30% to any CAZy database entry) and only 1.92% were more than 75% identical with proteins in NCBI environmental database, demonstrating that they have not been observed in previous metagenome projects. Phylogenetic analysis showed that APACMC harbored a broad range of pectinolytic bacteria and many of them were previously unrecognized.

Conclusions: The immensely diverse pectinolytic microbes and enzymes found in our study will expand the arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. Our study provides a powerful approach for targeted mining microbes and enzymes in numerous industries.

Keywords: Compost habitat; Lignocellulosic biofuel; Metagenomic; Pectin; Pectinolytic microbes and enzymes.

Fig. 1

A pipeline of metagenomics sequence-guided strategy coupled with enrichment culture technique used in this study

Fig. 2

The changes of physicochemical properties and bacterial communities during composting (a temperature; b water content and pH; c at the phylum level; d at the genus level)

Fig. 3

Taxonomy composition (a) and phylogenetic tree (b) of APACMC metagenome

Fig. 4

COG (a) and KEGG (b) functional categories of APACMC metagenome

Fig. 5

Phylogenetic distributions of CAZymes in the most abundant members in APACMC (a at phylum level; b at genus level; c at species level)

Fig. 6

Schematic representation of pectin structure (Schematic representation of pectin structure was modified from [19]) and the phylogenetic affiliation (The phylogenetic affiliations of key pectinolytic enzymes in APACMC were visualized by MEGAN6) of key pectinolytic enzymes in APACMC

Fig. 7

Similarity distribution of putative pectinolytic candidates (n = 1756) containing a catalytic domain (CD) or a carbohydrate-binding module (CBM) associated with pectinolytic activity. Sequences were compared to the NCBI-NR (red 1756 hits), CAZy (black 1464 hits), NCBI-ENV (blue 1560 hits), and Swiss-Prot (pink 927 hits) databases (best BLAST hit, E value ≤1e⁻⁵); 26 genes contained both a CD and CBM, whereas 1498 and 232 genes contained only a CD or CBM, respectively