3-46: Combinatorial engineering of endoglucanases using a cell-free protein expession system

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.