Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries

Abstract

SummaryA high‐throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester‐hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′‐O‐tri‐acetyluridine and 2′,3′,5′‐O‐tri‐hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…Metagenomic libraries used for functional selection of amidohydrolases were constructed from different soil DNA samples as described earlier [12]. The DNA fragment length varied from 5 to 17 kb in the different libraries.…”

“…Therefore, 7 plasmids were chosen for further analysis. N 4 -benzoylcytidine (12), instead of N 4 -benzoyl-2'-deoxycytidine (4), was also tested as a substrate-all 7 E. coli DH10B ∆pyrFEC::Km transformants harbouring the selected plasmids used the N 4 -benzoylcytidine (12) as a uridine source. The necessity of the auxiliary enzyme for successful selection was demonstrated by deletion of the E. coli cdd gene, which encodes cytidine deaminase.…”

“…The hydrolytic activity of the recombinant enzymes was assayed qualitatively by testing 29 different substrates ( Figure 4A): N 4 -acylated (2'-deoxy)cytidines and cytosines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16); N 2 -acetylisocytosines (17); chromogenic substrates such as p-nitroacetanilide (18) and p-nitrobenzanilide (19); various p-nitrophenyl (pNP) esters (20)(21)(22)(23)(24); terephthalate derivatives bis(2-hydroxyethyl) terephthalate (26), dimethyl terephthalate (27), and monomethyl terephthalate (28); and the beta-lactam nitrocefin (29). Hydrolysis of chromogenic substrates (18)(19)(20)(21)(22)(23)(24)(25) was analysed spectrophotometrically at 405 nm, and that of other substrates was analysed spectrophotometrically at 240-320 nm and using TLC or HPLC-MS ( Figures S4-S10).…”

“…Keeping in mind that the functions of 30%-40% of the genes in genomes remain unknown, the detection of new biocatalysts in the sequenced genomes can be difficult if no significant homology with known enzymes is observed [6,7]. 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].…”

“…Recently, it has been shown that a uridine auxotrophic strain of Escherichia coli can be successfully used for the identification of certain enzymes involved in the catabolism of modified heterocyclic bases or nucleosides [10,11,27] and for functional screening of esterases [12], when the appropriate uracil or uridine derivatives serve as substrates for the enzymes. Here, we present a method based on uridine auxotrophic E. coli, tailored especially for the selection of amidohydrolases from metagenomes.…”