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

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


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.

Original languageEnglish
Pages (from-to)1306-1315
Number of pages10
JournalChemistry and Biology
Issue number12
StatePublished - Dec 22 2010


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