Monday, April 19, 2010
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Combinatorial engineering of endoglucanases using a cell-free protein expession system

Tae-Wan Kim, Harshal A. Chokhawala, Harvey W. Blanch, and Douglas S. Clark. Energy Biosciences Institute, University of California Berkeley, 130 Calvin Hall, Berkeley, CA 94720

Bacteria and yeast have previously been employed as expression hosts for cellulase production; however, expressing cellulases by conventional cell-based approaches is time-consuming and often leads to inactive forms or low yields. These limitations have been a major roadblock in cellulase engineering for the production of biofuels. Herein we describe a cell-free expression platform using E. coli cell extract with overexpressed chaperones, which yields active cellulases in high yields in a high-throughput fashion. A library of chimeric endoglucanases combining catalytic domains and carbohydrate-binding modules was constructed using overlap extension PCR and successfully expressed using the cell-free expression method. The endoglucanase library was screened for hydrolytic activity against industrially relevant lignocellulosic substrates, including ionic-liquid pretreated Miscanthus and AFEX-pretreated corn stover. CBM fusions afforded increased activity for many enzymes toward crystalline cellulose. The high-throughput cell-free expression platform can thus provide insights into the relationship between CBM, CD, linker, and catalytic activity for any substrate of interest, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production.

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