The use of fosmid metagenomic libraries in preliminary screening for various biological activities

Abstract

BackgroundIt is generally believed that there are many natural sources of as yet unknown bioactive compounds with a high biotechnological potential. However, the common method based on the use of cell extracts in the preliminary screening for particular molecules or activities is problematic as amounts of obtained compounds may be low, and such experiments are hardly reproducible. Therefore, the aim of this work was to test whether a novel strategy to search for previously unknown biological activities can be efficient. This strategy is based on construction of metagenomic libraries and employment of Escherichia coli strains as cell factories producing compounds of properties potentially useful in biotechnology.ResultsThree cyanobacterial metagenomic libraries were constructed in the fosmid system. The libraries were screened for various biological activities. Extracts from selected E. coli clones bearing constructs with fragments of cyanobacterial genomes revealed antimicrobial or anticancer activities. Interestingly, stimulation of growth of host bacteria bearing particular plasmids with certain cyanobacterial genes was detected, suggesting a potential possibility for improvement of E. coli cultivation during biotechnological production. The most interesting plasmids were sequenced, and putative mechanisms of biological effects caused by cyanobacterial gene products are discussed.ConclusionsThe strategy of exploring cyanobacteria as sources of bioactive compounds, based on E. coli cell factories producing compounds due to expression of genes from metagenomic libraries, appears to be effective.

Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors

“…Normally, PKS type I and NRPS's pathways are organized in large assembly operons going from 20 kb to 100 kb in length [ 43 ]. Accordingly, several examples in the literature show that metagenomics has been successfully applied for the identification of novel pathways coding for bioactive compounds in diverse environments [ 44 – 49 ]. Although the functional-based approach allows obtaining completely original genes in an independent-sequence way, numerous studies using next-generation sequencing techniques and subsequent bioinformatic mining of the metagenomes have also achieved novel gene clusters involved in secondary metabolites production [ 50 – 53 ].…”

The most widespread problems in the function-based microbial metagenomics

“…Adaptation of professional equipment in the combination with high-throughput screening including fluorophores and chromophores such as nitrophenoles, umbelliferones, fluoresceins, rhodamines and BODIPY dyes, could be the solution to improve exploration of the metagenomic library clones while maintaining the selectivity and sensitivity of the assay method (Reymond et al, 2009). Felczykowska et al (2014) tested individual clones belonging to a fosmid metagenomic library made from eDNA isolated from cyanobacteria bloom obtained in the Baltic Sea. Approximately 400 E. coli clones bearing environmental DNA were screened for anticancer and antibacterial activity.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…Most agar-plate-based methods suffer from low sensitivity due to the diffusion of the soluble products. Crude-cell-lysate-based methods usually have low throughput and are less reproducible (Felczykowska, Dydecka et al 2014). Lipases/esterases constitute a major fraction of enzymes derived from metagenomic screening simply due to the availability of easy high-throughput screening methods (Taupp, Mewis et al 2011, Reyes-Duarte, Ferrer et al 2012.…”