Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library

Shaun M. Lippow, Tae Seok Moon, Subhayu Basu, Sang-Hwal Yoon, Xiazhen Li, Brad A. Chapman, Keith Robison, Daša Lipovšek, Kristala L.J. Prather
2010 Chemistry and Biology  
Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which interresidue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase
more » ... nzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways. X XX 383 406 441 463 X X X 290 AarI
doi:10.1016/j.chembiol.2010.10.012 pmid:21168766 fatcat:2mbqudqofzcvfhmjim52p7ngle