High-throughput selection for cellulase catalysts using chemical complementation.
Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.
Peralta-Yahya
Pamela
P
Department of Chemistry, Columbia University, New York, New York 10027, USA.
Carter
Brian T
BT
Lin
Hening
H
Tao
Haiyan
H
Cornish
Virginia W
VW
D000818:Animals; D044822:Biodiversity; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D016923:Cell Death; D002470:Cell Survival; D002480:Cellulase / Q000737:chemistry*; D002621:Chemistry / Q000379:methods*; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D006031:Glycosylation; D006867:Hydrolases / Q000737:chemistry; D008956:Models, Chemical; D016296:Mutagenesis
D004247:DNA; D006867:Hydrolases; D002480:Cellulase