Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

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Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data ... continued below

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Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D'haeseleer, P.; Lowry, S. et al. November 15, 2009.

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Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

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  • Journal Name: PLoS One; Related Information: Journal Publication Date: 2010

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  • Report No.: LBNL-3058E
  • Grant Number: DE-AC02-05CH11231
  • Office of Scientific & Technical Information Report Number: 981736
  • Archival Resource Key: ark:/67531/metadc1013625

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  • November 15, 2009

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  • Oct. 14, 2017, 8:36 a.m.

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  • Oct. 17, 2017, 7 p.m.

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Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D'haeseleer, P.; Lowry, S. et al. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community, article, November 15, 2009; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1013625/: accessed October 18, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.