Engineering enzyme specificity using computational design of a defined-sequence library |
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Authors: | Lippow Shaun M Moon Tae Seok Basu Subhayu Yoon Sang-Hwal Li Xiazhen Chapman Brad A Robison Keith Lipov?ek Da?a Prather Kristala L J |
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Institution: | Codon Devices, Inc., 99 Erie Street, Cambridge, MA 02139, USA. slippow@gmail.com |
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Abstract: | 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. |
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