Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome by Synthetic Metagenomics

Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes; β-glucosidase, α-L-arabinofuranosidase, β-xylosidase, and an endo-1,4-β-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation, and phylogenetic trees were created to depict relatedness to known enzymes. The candidate synthesized genes were cloned into the pET26b(+) plasmid to enable inducible expression in Escherichia coli (E. coli). Each candidate gene was cloned as a fusion protein bearing an amino-terminal PelB motif required for periplasmic localization and subsequent secretion, and a carboxy-terminal hexahistidine (6xHIS) tag to enable affinity purification. We demonstrated IPTG-inducible accumulation of all four fusion proteins. The putative β-glucosidase fusion protein was efficiently secreted but did not permit E. coli to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl β-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). By contrast, the affinity purified putative endo-1,4-β-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate over this same pH range, with maximal activity at pH 7. At this optimal pH, KM , Vmax, and kcat were determined to be 32.005 +/- 4.72 μM, 1.16x10-5 +/- 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our synthetic metagenomic 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.