Recovery and functional validation of hidden soil enzymes in metagenomic libraries

Abstract

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Keywords: Environmental microbiology; Functional genomics; Metagenomics; Microbial genomics.

Figure 1

Pipeline overview. Isolated metagenomic clones are pooled in one sample for a massive sequencing analysis and independently analyzed by Sanger sequencing, in order to map the metagenomic inserts to their corresponding bacterial clones. After DNA assembly and clone assignment processes, ORF predictions and functional characterization of predicted putative proteins (e.g., PP1 and PP2) are performed. Selected coding sequences (e.g., CDS1 and CDS2) associated with the enzymatic activities of interest are matched to the original metagenomic clones or subcloned for independent maintenance in plasmid vectors. Finally, functional analyses on subclones expressing the predicted proteins allow the recovery of several enzymatic activities not identified in traditional functional metagenomic assays

Figure 2

Gene Ontology functions of the annotated fraction of the metagenome. Proteins with associated PFAM domains were mapped to Gene Ontology terms (GOSlim). Most of the terms are associated with energy metabolism and transport in and out of the cell. Proteins can be binned into more than one category and therefore the total number of annotations is higher than the total number of proteins

Figure 3

Bacterial enzymatic activity. (a) Proteolytic activity determination by the colorimetric method of Folin Ciocalteu reagent using casein as substrate of the reaction together with bacterial extracts from E. coli EPI300 metagenomic clones F8_C18 (harboring Prot1 CDS) or F38_C21 (harboring Prot2 CDS). (b) Lipolytic activity detection by p‐Nitrophenyl butyrate degradation of the bacterial extract derived from E. coli EPI300 metagenomic clone F5_C17 (harboring LipM CDS). In (a) and (b) E. coli EPI300 was used as a negative control of the enzymatic activities. (c) Proteolytic activity determination of bacterial extracts derived from E. coli LMG‐194 clones harboring either pBAD_Prot1 or pBAD_Prot2. (D) Lipolytic activity detection of the bacterial extract derived from E. coli BL21 harboring pET100_LipM plasmid. In (c) and (d), the respective nontransformed E. coli strains were used as negative controls of enzymatic activity. Error values represent standard deviations from three replicates in each case. *Indicates a significant difference in the proteolytic activity from clones pBAD_Prot1 and pBAD_Prot2 (p‐value < .05) compared with negative control. **Indicates a significant difference in the lipolytic activity of the bacterial extract derived from clone pET100_LipM (p‐value < .05) compared with negative control

Figure 4

Partial protease characterization. (a) Effect of temperature on protease activities of Prot1 and Prot2. (b) Effect of pH on protease activities of Prot1 and Prot2. (c) Effect of metal ions and inhibitor (EDTA) on the enzymatic activities of Prot1 and Prot2