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

Mackenzie Thornbury, Jacob Sicheri, Caroline Guinard, David Mahoney, Francis Routledge, Matthew Curry, Mariam Elaghil, Nicholas Boudreau, Angela Tsai, Patrick Slaine, Emma Finlayson-Trick, Landon Getz, Jamie Cook, John Rohde, View ORCID ProfileCraig McCormick
doi: https://doi.org/10.1101/288985
Mackenzie Thornbury
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Jacob Sicheri
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Caroline Guinard
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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David Mahoney
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Francis Routledge
2Department of Neuroscience, Dalhousie University, Nova Scotia, Canada
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Matthew Curry
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Mariam Elaghil
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Nicholas Boudreau
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Angela Tsai
3Faculty of Science, Dalhousie University, Nova Scotia, Canada
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Patrick Slaine
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Emma Finlayson-Trick
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Landon Getz
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Jamie Cook
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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John Rohde
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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Craig McCormick
1Department of Microbiology and Immunology, Dalhousie University, Nova Scotia, Canada
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  • ORCID record for Craig McCormick
  • For correspondence: craig.mccormick@dal.ca
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Abstract

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components 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 novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a β-xylanase, endoxylanase, β-glucosidase, and an ⍺-L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative β-xylanase into the pET26b(+) plasmid, enabling inducible gene expression in Escherichia coli (E. coli) and periplasmic localization. We demonstrated IPTG-inducible accumulation of β-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative β-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway into E. coli to enable biofuel production from wood pulp feedstock.

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Posted March 27, 2018.
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Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome
Mackenzie Thornbury, Jacob Sicheri, Caroline Guinard, David Mahoney, Francis Routledge, Matthew Curry, Mariam Elaghil, Nicholas Boudreau, Angela Tsai, Patrick Slaine, Emma Finlayson-Trick, Landon Getz, Jamie Cook, John Rohde, Craig McCormick
bioRxiv 288985; doi: https://doi.org/10.1101/288985
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Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome
Mackenzie Thornbury, Jacob Sicheri, Caroline Guinard, David Mahoney, Francis Routledge, Matthew Curry, Mariam Elaghil, Nicholas Boudreau, Angela Tsai, Patrick Slaine, Emma Finlayson-Trick, Landon Getz, Jamie Cook, John Rohde, Craig McCormick
bioRxiv 288985; doi: https://doi.org/10.1101/288985

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