TY - JOUR T1 - Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome by Synthetic Metagenomics JF - bioRxiv DO - 10.1101/288985 SP - 288985 AU - Mackenzie Thornbury AU - Jacob Sicheri AU - Patrick Slaine AU - Landon J. Getz AU - Emma Finlayson-Trick AU - Jamie Cook AU - Caroline Guinard AU - Nicholas Boudreau AU - David Jakeman AU - John Rohde AU - Craig McCormick Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/07/18/288985.abstract N2 - 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, β-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 μΜ, 1.16×10-5 ± 3.55×10-7 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.Author summary Plants depend on a mixture of complex polysaccharides including cellulose, hemicellulose and lignin to provide structural support. Microorganisms use enzymes to break down these polysaccharides into simple sugars like glucose that they burn for energy. These microbial enzymes have the potential to be repurposed for industrial biofuel production. Previously, we showed that bacteria in the porcupine gut produce enzymes that break down cellulose and hemicellulose. Here we report the identification and synthesis of four genes from the porcupine microbiome that encode enzymes that can break down complex plant polysaccharides, including a β-glucosidase, an β-L-arabinofuranosidase, a β-xylosidase, and an endo-1,4-β-xylanase. These genes were introduced into the model bacterium Escherichia coli (E coli) to test the properties of their respective gene products. All four enzymes accumulated following the induction of gene expression, but only the putative cellulose-degrading β-glucosidase and hemicellulose-degrading β-xylosidase and β-L-arabinofuranosidase enzymes were efficiently secreted out of the bacteria where they could access their target polysaccharides. Expression of the putative β-glucosidase did not allow E. coli to grow on cellobiose as a sole carbon source, and the purified enzyme failed to cleave p-NPG in vitro. By contrast, the purified putative endo-1,4-β-xylanase cleaved an artificial hemicellulose substrate in vitro across a range of acidic and neutral pH values, with maximal activity at pH 7. This study demonstrates the power of our approach to identify novel microbial genes that may be useful for industrial biofuel production. ER -