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

• Main Authors: Lippow, Shaun M. (Author), Moon, Tae Seok (Contributor), Basu, Subhayu (Author), Yoon, Sang-Hwal (Contributor), Li, Xiazhen (Author), Chapman, Brad A. (Author), Robison, Keith (Author), Lipovšek, Daša (Author), Prather, Kristala L. Jones (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Chemical Engineering (Contributor), Massachusetts Institute of Technology. Synthetic Biology Center (Contributor)
• Format: Article
• Language: English
• Published: Elsevier, 2015-03-17T16:10:57Z.
• Subjects: Article
• Online Access: Get fulltext

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.
United States. Office of Naval Research. Young Investigator Program (Grant N000140510656)
National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center. Grant EEC-0540879)
MIT Faculty Start-up Fund
Codon Devices, Inc.