Journal
CHEMISTRY & BIOLOGY
Volume 17, Issue 12, Pages 1306-1315Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2010.10.012
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Funding
- Codon Devices, Inc.
- Office of Naval Research [N000140510656]
- National Science Foundation through the Synthetic Biology Engineering Research Center [EEC-0540879]
- MIT
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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.
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