title: Engineering enzyme specificity using computational design of a defined-sequence library.
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.
authors.affiliation: Codon Devices, Inc., 99 Erie Street, Cambridge, MA 02139, USA. slippow@gmail.com
authors.forename: Shaun M
authors.initials: SM
authors.lastname: Lippow
authors.forename: Tae Seok
authors.initials: TS
authors.lastname: Moon
authors.forename: Subhayu
authors.initials: S
authors.lastname: Basu
authors.forename: Sang-Hwal
authors.initials: SH
authors.lastname: Yoon
authors.forename: Xiazhen
authors.initials: X
authors.lastname: Li
authors.forename: Brad A
authors.initials: BA
authors.lastname: Chapman
authors.forename: Keith
authors.initials: K
authors.lastname: Robison
authors.forename: Daša
authors.initials: D
authors.lastname: Lipovšek
authors.forename: Kristala L J
authors.initials: KL
authors.lastname: Prather
mesh_terms: D000595:Amino Acid Sequence; D019295:Computational Biology; D015723:Gene Library; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D005949:Glucose Oxidase