Abstract
In this study, the development of a rapid, high-throughput method for the selection of amide-hydrolysing enzymes from the metagenome is described. This method is based on uridine auxotrophic Escherichia coli strain DH10B ∆pyrFEC and the use of N4-benzoyl-2’-deoxycytidine as a sole source of uridine in the minimal microbial M9 medium. The approach described here permits the selection of unique biocatalysts, e.g., a novel amidohydrolase from the activating signal cointegrator homology (ASCH) family and a polyethylene terephthalate hydrolase (PETase)-related enzyme.
Highlights
It is estimated that the Earth is inhabited by 1011 –1012 microbial species, of which only ~104 have been cultured in the laboratory [1]
Amidases are used for the biosynthesis
Metagenomic libraries used for functional selection of amidohydrolases were constructed from different soil DNA samples as described earlier [12]
Summary
It is estimated that the Earth is inhabited by 1011 –1012 microbial species, of which only ~104 have been cultured in the laboratory [1]. Metagenomics, the improvement of high-throughput screening methods, and the development of host expression systems for metagenome-derived genes, systems biology, and gene synthesis may together open the gateway to the useful information that hides in unexplored genetic resources, e.g., new enzymes with unique properties and novel scaffolds applicable for evolution in vitro [8,9,10,11,12]. The amidohydrolases/amidases are large classes (EC 3.5.1 and 3.5.2.) of enzymes which hydrolyse a wide variety of linear and cyclic amides. These enzymes are involved in several metabolic pathways, e.g., amide digestion and absorption by breaking carbon and nitrogen bonds, prokaryotic amide degradation, and synthesis of many important eukaryotic growth factors.
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