Functional Screening of Metagenomic Libraries: Enzymes Acting on Greasy Molecules as Study Case

Martínez-Martínez, Mónica; Golyshin, Peter N.; Ferrer, Manuel

doi:10.1007/8623_2015_104

Cited by 1 publication

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“…In this sense, functional metagenomics has arisen as a powerful tool (Mirete et al, 2016), since it allows, on one hand, the discovery of novel biocatalysts of uncultured bacteria, thus circumventing traditional methods that rely on cultivation, and on the other hand, avoids the major drawback of sequence-based screening technologies, which do not allow direct conclusions about the functionality and biochemical parameters of the encoded enzymes. Nevertheless, functional metagenomics has the important limitation of setting up function-driven screening assays (Martínez-Martínez et al, 2015), which in most cases are very tedious and time-consuming, requiring complex substrates and sophisticated chromogenic assays as well as high-performance liquid chromatography (HPLC) or similar analytical methods. Thus, it is not surprising that the majority of metagenome-derived enzymes that have been biochemically characterized are mainly esterases/lipases and glycoside hydrolases (Lopez-Lopez et al, 2014; Sathya and Khan, 2014; Ufarte et al, 2015).…”

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confidence: 99%

Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries

et al. 2016

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Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

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