Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library
Author(s)Lippow, Shaun M.; Moon, Tae Seok; Basu, Subhayu; Yoon, Sang-Hwal; Li, Xiazhen; Chapman, Brad A.; Robison, Keith; Lipovšek, Daša; Prather, Kristala L. Jones; ... Show more Show less
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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 inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme 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.
DepartmentMassachusetts Institute of Technology. Department of Chemical Engineering; Massachusetts Institute of Technology. Synthetic Biology Center
Chemistry and Biology
Lippow, Shaun M., Tae Seok Moon, Subhayu Basu, Sang-Hwal Yoon, Xiazhen Li, Brad A. Chapman, Keith Robison, Daša Lipovšek, and Kristala L.J. Prather. “Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library.” Chemistry & Biology 17, no. 12 (December 2010): 1306–1315. © 2010 Elsevier Ltd.
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