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

PloS One
Martin AllgaierPhilip Hugenholtz

Abstract

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, approximately 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 deg...Continue Reading

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Datasets Mentioned

BETA
ID
41493
SRA010300.1
GU178033
GU178768

Methods Mentioned

BETA
PCR

Software Mentioned

ClustalX
Newbler assembler
KaleidaGraph
BLASTX
R
RAST
ARB
Newbler
SEED
ade4

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