TY - JOUR
T1 - Engineering enzyme specificity using computational design of a defined-sequence library
AU - Lippow, Shaun M.
AU - Moon, Tae Seok
AU - Basu, Subhayu
AU - Yoon, Sang Hwal
AU - Li, Xiazhen
AU - Chapman, Brad A.
AU - Robison, Keith
AU - Lipovšek, Daša
AU - Prather, Kristala L.J.
N1 - Funding Information:
Work performed at Codon Devices, Inc., was funded by the Codon Devices, Inc., research budget. Work performed at MIT was supported by grants from the Office of Naval Research Young Investigator Program (Grant No. N000140510656) and the National Science Foundation through the Synthetic Biology Engineering Research Center (Grant No. EEC-0540879), as well as with start-up funds provided by MIT to K.L.J.P.
PY - 2010/12/22
Y1 - 2010/12/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=78650471329&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2010.10.012
DO - 10.1016/j.chembiol.2010.10.012
M3 - Article
C2 - 21168766
AN - SCOPUS:78650471329
SN - 1074-5521
VL - 17
SP - 1306
EP - 1315
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 12
ER -