Characterization of novel lignocellulose-degrading enzymes from the porcupine microbiome using synthetic metagenomics.
Thornbury M, Sicheri J, Slaine P, Getz LJ, Finlayson-Trick E, Cook J, Guinard C, Boudreau N, Jakeman D, Rohde J, McCormick C
PLoS One. 2019. doi: 10.1371/journal.pone.0209221
COMMENT: This work combines metagenomics and synthetic biology strategies to obtain new enzymes with lignocellulose-degrading capacity. First, a shotgun metagenomics using de novo assembly approach from porcupine fecal DNA samples allowed the selection a set of 4 candidates with possible lignocellulose-degrading activity. These candidate genes were synthesized and cloned into plasmid expression vectors. Activity tests for the 4 synthetic enzymes were carried out and, finally, a 1,4-β-xylanase protein was selected by its capacity of cleavage of a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7.
The authors summarize the bioinformatics pipeline of this work here:
The bioinformatic pipeline began with Illumina MiSeq data previously collected from a porcupine fecal DNA sequencing project. Reads were checked for quality and trimmed, concatemerized via MegaHit, and open reading frames were identified using Prodigal. Protein sequences of interest were identified by pHMMER using various protein databases and were selected for matches of interest based on e-value selection.
Our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases