Directed evolution of a glycosynthase via chemical complementation.
Recently, we reported a general assay for enzyme catalysis based on the yeast three-hybrid assay, Chemical Complementation, which is intended to expand the range of chemical reactions to which directed evolution can be applied. Here, Chemical Complementation was applied to a glycosynthase derived from a retaining glycosidase, an important class of enzymes for carbohydrate synthesis. Using the yeast three-hybrid assay, the glycosynthase activity of the E197A mutant of the Cel7B from Humicola insolens was linked to transcription of a LEU2 reporter gene, making cell growth dependent on glycosynthase activity in the absence of leucine. Then the LEU2 selection was used to isolate the most active glycosynthase from a Glu197 saturation library, yielding an E197S Cel7B variant with a 5-fold increase in glycosynthase activity. These results not only establish Chemical Complementation as a platform for the directed evolution of glycosynthases, but also show the generality of this approach and the ease with which it can be applied to new chemical reactions.
Lin
Hening
H
Department of Chemistry, Columbia University, New York, New York 10027, USA.
Tao
Haiyan
H
Cornish
Virginia W
VW
D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods; D007930:Leucine / Q000378:metabolism; D017354:Point Mutation; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
C056374:endoglucanase 1; D002480:Cellulase; D007930:Leucine