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

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.

Cytidine deaminases catalyze the conversion of N ( S , O ) 4 -substituted pyrimidine nucleosides

“…Three metagenomic clones (EH, F14, and F18) were selected using a previously described method with uridine auxotrophic E. coli DH10B Δ pyrFEC ::Km ( 23 ) cells and N 4 -benzoyl-2′-deoxycytidine ( 25 ) as a 2′-deoxyuridine ( 1 ) source ( 21 ). Unexpectedly, the sequence analysis of the DNA fragments from these clones failed to identify any of the typical amidohydrolase-encoding genes but showed the presence of open reading frames (ORFs) with high 69 to 85% sequence identity to CDAs found in the National Center for Biotechnology Information GenBank database (table S1) ( 24 ).…”

“…PCR fragments were ligated at 4°C for 18 hours, and after incubation, DNA plasmids of site-directed mutants were transformed into DH5α E. coli cells and plated on LB agar plates supplemented with ampicillin (100 μg/ml). The functional screening of active random mutants was selected by using E. coli HMS174 Δ pyrF ∆ cdd strain and N 4 -benzoyl-2′-deoxycytidine as a 2′-deoxyuridine source ( 21 ). Isopropyl-β- d -thiogalactopyranoside (1 mM) was added to a selective medium for induction of gene expression.…”

“…In this study, a functional metagenomic mining using complementation of uridine auxotrophy and N 4 -benzoyl-2′-deoxycytidine (25) as a source of uridine (21) has led to the successful isolation of several CDA homologs. Here, we show that CDAs convert different N 4 -acyl-arylpyrimidine, N 4 -/S 4 -/O 4 -alkyl-arylpyrimidine, and N 4 -/ S 4 -/O 4 -arylpyrimidine nucleosides, including capecitabine, directly into derivatives of uridine.…”

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases

“…Cells carrying pET21b-yqfB were used as a positive control, while cells transformed with the pET21b vector without an insert were used as a negative control. The ability of a uridine auxotrophic strain to grow on ac4C supplemented media was an indicator of the amidohydrolytic activity of the target protein [ 19 ].…”

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs

“…Both of these enzymes are unique monomeric amidohydrolases belonging to human activating signal cointegrator homology (ASCH) domain-containing proteins, which are widespread and diverse, but so far most of them do not have a confirmed function [ 18 ]. Although both YqfB and D8_RL can catalyze the conversion of N 4 -acylated cytidines, their structural and catalytic properties are different [ 16 ], which makes both enzymes equally interesting objects for this study.…”