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.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library

“…This study demonstrates a bioinformatic pipeline for complete metagenomic library sequencing and mining for BGC-containing clones, providing full annotation of inserts prior to targeted heterologous expression. Attempts to leverage NGS capabilities to sequence collections of metagenomic clones have so far relied on a hybrid approach, for example using “tags” for each clone that are created via amplification and Sanger sequencing of individual clones, then pooling the clones and performing sequencing using NGS ( Dzunkova et al, 2012 ; Calderon et al, 2019 ). In contrast, this study presents a strategy capable of directly sequencing large numbers of clones, while maintaining insert positional information within the library, a critical step in targeting specific cloned BGCs for conjugal transfer to a compatible heterologous host and functional screening for bioactive secondary metabolites.…”

Metagenomic screening strategies for bioprospecting enzymes from environmental samples

“…Proteases are the biocatalysts that hydrolyze the peptide bonds which join the amino acid in chains. These are continuously utilized in pharmaceutical, food, detergent, and beverage industries [50]. There is a wide spectrum of protease sources available, which include plants, animals as well as microorganisms.…”

Potential Use of Microbial Community Genomes in Various Dimensions of Agriculture Productivity and Its Management: A Review

“…In a different survey, a novel salt-tolerant, chitobiosidase was derived from chitin-amended disease-suppressive agricultural soil with the use of a metagenomic library ( Cretoiu et al, 2015 ). Rhizospheric soil samples collected from three different Solanum phureja farms located in the Cundinamarca Andean Plateau, Colombia revealed unique lipase, esterase, and protease enzymes that have a biotechnological application ( Calderon et al, 2019 ).…”